Objective: To investigate the effect of Zuogui Jiangtang Jieyu Formula (左归降糖解郁方, ZJJF) on hippocampal neuron apoptosis in diabetic rats with depression and to ascertain whether its mechanism involves the regulation of JNK signaling pathway. Methods: (i) A total of 72 specific pathogen-free (SPF) grade male Sprague Dawley (SD) rats were randomly divided into six groups, with 12 rats in each group: control, model, metformin (Met, 0.18 g/kg) + fluoxetine (Flu, 1.8 mg/kg), and the high-, medium-, and low-ZJJF dosages (ZJJF-H, 20.52 g/kg; ZJJF-M, 10.26 g/kg; ZJJF-L, 5.13 g/kg) groups. All groups except control group were injected once via the tail vein with streptozotocin (STZ, 38 mg/kg) combined with 28 d of chronic unpredictable mild stress (CUMS) to establish diabetic rat models with depression. During the CUMS modeling period, treatments were administered via gavage, with control and model groups receiving an equivalent volume of distilled water for 28 d. The efficacy of ZJJF in reducing blood sugar and alleviating depression was evaluated by measuring fasting blood glucose, insulin, and glycated hemoglobin levels, along with behavioral assessments, including the open field test (OFT), forced swim test (FST), and sucrose preference test (SPT). Hippocampal tissue damage and neuronal apoptosis were evaluated using hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) staining. Apoptosis-related proteins Bax, Bcl-2, caspase-3, and the expression levels of JNK/Elk-1/c-fos signaling pathway were detected using Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). (ii) To further elucidate the role of JNK signaling pathway in hippocampal neuronal apoptosis and the pharmacological effects of ZJJF, an additional 50 SPF grade male SD rats were randomly divided into five groups, with 10 rats in each group: control, model, SP600125 (SP6, a JNK antagonist, 10 mg/kg), ZJJF (20.52 g/kg), and ZJJF (20.52 g/kg) + Anisomycin (Aniso, a JNK agonist, 15 mg/kg) groups. Except for control group, all groups were established as diabetic rat models with depression, and treatments were administered via gavage for ZJJF and intraperitoneal injection for SP6 and Aniso for 28 d during the CUMS modeling period. Behavioral changes in rats were evaluated through the OFT, FST, and SPT, and hippocampal neuron damage and apoptosis were observed using HE staining, Nissl staining, TUNEL staining, and transmission electron microscopy (TEM). Changes in apoptosis-related proteins and JNK signaling pathway in the hippocampal tissues of rats were also analyzed. Result: (i) ZJJF significantly reduced the high blood glucose, insulin, and glycated hemoglobin levels in model rats (P < 0.01). It increased autonomous activity and decreased despair-like behaviors (P < 0.01), improved the pathological damage of hippocampal neurons,increased the number of neuronal nuclei (P < 0.01), and red uced the number of mechanocytes, vacuolar cells, and apoptotic neurons (P < 0.05, P < 0.01, and P < 0.01, respectively). ZJJF down-regulated the expression levels of pro-apoptotic proteins Bax and caspase-3 (P < 0.01), up-regulated the anti-apoptotic protein Bcl-2 (P < 0.01), and significantly inhibited the overexpression of phosphorylated JNK (p-JNK), Elk-1, and c-fos (P < 0.01). (ii) SP6 increased autonomous activity and reduced despair time in model rats (P < 0.05), although it had no significant effects on sucrose preference (P > 0.05). It increased the number of Nissl bodies in hippocampal neurons (P < 0.01), reduced the protein expression levels of Bax (P < 0.01) and caspase-3 (P < 0.05), and decreased the number of apoptotic neurons (P < 0.05). SP6 also increased the expression level of Bcl-2 (P < 0.01), and inhibited the high expression levels of p-JNK, Elk-1, and c-fos (P < 0.01, P < 0.01, and P < 0.05, respectively), suggesting that hippocampal neuronal apoptosis in diabetic rats with depression is associated with abnormal activation of JNK signaling pathway. Compared with ZJJF group, ZJJF + Aniso group showed a decrease in sucrose preference (P < 0.05) and an increase in despair time (P < 0.01) with more notable hippocampal neuronal damage. This group also exhibited a decrease in expression level (P < 0.01) Bcl-2 and an increase in expression levels of Bax, caspase-3, p-JNK, Elk-1, and c-fos (P < 0.01, P < 0.05, P < 0.05, P < 0.01, and P < 0.05, respectively), indicating that the antidepressant effects of ZJJF, its improvement of neuronal apoptosis, and regulation of JNK signaling molecules could all be reversed by a specific JNK agonist. Conclusion ZJJF exerts a significant hypoglycemic effect and ameliorates the apoptosis of hippocampal neurons by inhibiting the activation of JNK signaling pathway, which is a promising formula for the treatment of diabetic depression in clinical settings.