ObjectiveTo investigate the synergistic inhibitory effect of chidamide （CHI） combined with compound Kushen injection （CKI） on breast cancer MCF-7 cells and to elucidate the potential molecular mechanisms based on transcriptomics analysis. MethodsCell proliferation was detected by the methyl thiazolyl tetrazolium （MTT） assay， and the "one belt and one line" method was adopted to screen the optimal mass concentration for synergistic effect. According to the combination index （CI） values， cells were allocated into the blank， CHI （1.6 mg·L^-1）， CKI （0.4 g·L^-1）， and CHI+CKI （1.6 mg·L^-1+0.4 g·L^-1） groups. Transwell assay was employed to examine cell migration， and flow cytometry to measure cell cycle distribution and apoptosis rate. Transcriptome sequencing （RNA-seq） and weighted gene co-expression network analysis （WGCNA） were performed to screen the key modules highly correlated with the cell inhibition rate. Candidate genes were identified by intersecting module genes with differentially expressed genes （DEGs）， followed by functional enrichment analysis. Western blot was employed to quantify the protein levels of histone deacetylase 1 （HDAC1）， protein kinase A （PKA）， cyclin-dependent kinase inhibitor 1 （p21）， cAMP response element-binding protein （CREB） and its phosphorylated form （p-CREB）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in cells of each group. ResultsCompared with the blank group， both CHI and CKI monotherapies inhibited the proliferation of MCF-7 cells in a mass concentration-dependent manner. Compared with the blank and monotherapy groups， CHI+CKI inhibited cell migration， increased the proportion of cells arrested in the G₀/G₁ phase， and elevated the apoptosis rate （P<0.01）. WGCNA identified the MEred module strongly correlated with the inhibition rate （r=0.90， P<0.01）， yielding 97 candidate genes upon intersection with DEGs. Kyoto Encyclopedia of Genes and Genomes （KEGG） analysis indicated significant enrichment of the cAMP signaling pathway. Western blot results showed that compared with the blank group， all treatment groups down-regulated the expression of HDAC1， up-regulated the expression of PKA and p21， and increased the p-CREB/CREB and Bax/Bcl-2 ratios （P<0.01）. Compared with the monotherapy groups， CHI+CKI further up-regulated the PKA and p21 expression and increased the p-CREB/CREB and Bax/Bcl-2 ratios （P<0.01）. ConclusionThe combination of CHI and CKI exerts a significant synergistic anti-tumor effect. CHI exerts epigenetic regulation by inhibiting HDAC1， which， in combination with CKI， may further contribute to the activation of the cAMP/PKA/CREB signaling pathway， leading to up-regulation of p21 expression and the Bax/Bcl-2 ratio， thereby promoting cell cycle arrest and apoptosis.