【Abstract】 Objective The present study aims to elucidate the effects and mechanisms of Biejiajian Pill (BJJP)-containing medicated serum on the malignant biological behaviors of hepatocellular carcinoma Huh7 cells. Methods CMTM6 (CKLF-like MARVEL transmembrane domain containing 6)-specific siRNA was used to knock down the expression of CMTM6. Normal rat serum and low (0.55 g/kg), medium (1.1 g/kg), and high (2.2 g/kg) BJJP-containing medicated sera were prepared from healthy SD rats. Huh7 cells were cultured with normal fetal bovine serum (BC), normal rat serum (NC) and low, medium and high doses of drug-containing serum of BJJP (LBJJP, MBJJP and HBJJP), respectively. BJJP-containing serum and si-CMTM6 were applied to Huh7 cancer cells, and the cell proliferation, migration and invasion abilities were evaluated by CCK-8 and Transwell asssays, respectively. Western blot was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), epithelial mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, Vimentin), and CMTM6. Results CMTM6 knockdown significantly reduced the mRNA and protein expression level of CMTM6 in Huh7 cells (P<0.05). No significant differences were observed between BC and NC groups in proliferation, migration, invasion, or expression levels of PCNA, EMT markers, and CMTM6 (all P>0.05). BJJP-containing serum treatment markedly inhibited Huh7 cell proliferation, migration, and invasion, downregulated PCNA, CMTM6, N-cadherin, and Vimentin expression, while upregulating E-cadherin compared to NC group (all P<0.05). CMTM6 knockdown suppressed malignant behaviors, with reduced PCNA, Vimentin, N-cadherin and elevated E-cadherin expression (all P<0.05). Conclusions BJJP-containing medicated serum significantly inhibits Huh7 cell growth, invasion, migration, and EMT progression, potentially mediated through CMTM6 suppression.