Objective: The present study was aimed to investigate the neuroprotective effect of Croton hirtus (CH) extract against streptozotocin (STZ) in rats. Methods: (i) The sub-chronic toxicity consisted of 24 adult rats of either sex weighing from 160 to 200 g were divided into four groups with six rats in each group. Rats in group 1 received Dimethyl sulfoxide (DMSO) mixed with saline; rats in groups 2, 3, and 4 received 100, 200, and 400 mg/kg of methanolic extract of CH (MECH) orally by gavage administration for 28 d, respectively. The functional observation battery and locomotor activity were graded as part of their neurobehavioral activity and the brain regions, including cortex and hippocampus, were analyzed for neuropathological abnormalities. (ii) The main research consisted of 30 adult male Wistar rats weighing from 160 to 200 g, which were divided into five groups and six rats in each group, including control (I), STZ (II), Donepezil (III), MECH (100 mg/kg, IV), and MECH (200 mg/kg, V) groups. Rats in group I received citrate buffer orally and DMSO mixed with saline for 14 d. Rats in group II received STZ via intracerebroventricular injection (3 mg/kg, bilateral ICV-STZ) on days 1 and 3 followed by DMSO mixed with saline for 14 d. Rats in groups III, IV, and V received STZ administration on days 1 and 3 followed by Donepezil [3 mg/(kg·d), p.o.] and MECH [100 and 200 mg/(kg·d), p.o.] for 14 d. Rats were tested for learning and memory parameters such as Morris water maze (MWM) and passive avoidance test (PAT). They were sacrificed after completing behavioural experiments; brains were harvested to estimate the acetylcholinesterase (AChE), lipid peroxidation (LPO), glutathione (GSH), and superoxide dismutase (SOD) by using UV-Visible Spectrophotometer; caspase-3 was evaluated by total fluorescence emission spectra; amyloid β (Aβ) levels were detected using enzyme-linked immuosorbent assay (ELISA); and histopathological examination was conducted in the CA1 region of the hippocampus. Results : (i) The sub-chronic toxicity results revealed that open field test parameters including line crossing, rearing, entering the middle square, defecating, or urinating did not differ significantly in the MECH rats (P > 0.05). The histopathological observation did not show any lesions in the neuronal cells and inflammation in both the regions in MECH rats compared with control rats. (ii) The main study findings demonstrated that STZ-treated rats showed asignificant increase in impairment in learning and memory parameters (P < 0.001), the levels of AChE, caspase-3, Aβ (1-40 and 1-42), and LPO were increased significantly (P < 0.001), and significant decrease was found in the levels of SOD (P < 0.001) and GSH (P < 0.01). Moreover, neuronal damage was found in the hippocampus. In contrast, STZ-induced behavioural and biochemical impairments in rats were considerably decreased by treatment with MECH dose-dependently. Conclusion MECH significantly prevented the memory deficit induced by STZ due to antioxidant action. Restoration of cholinergic functioning may be the cause of behavioural improvement. Therefore, MECH may be able to treat cognitive disorders like Alzheimer's disease (AD).