Objective To elucidate the effects and mechanisms of Biejiajian pill (BJJP)-containing serum on the malignant biological behaviors of hepatocellular carcinoma Huh7 cells. Methods This research knocked down CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) expression using a CMTM6-specific small interfering RNA (siRNA). Healthy Sprague-Dawley rats were used to prepare normal rat serum and low- (0.55 g/ kg), medium- (1.1 g/kg), and high- (2.2 g/kg) BJJP-containing. Huh7 cells were cultured with normal fetal bovine serum (BC), normal rat serum (NC), and low-, medium-, and high-dose BJJP serum (LBJJP, MBJJP, and HBJJP, respectively). BJJP-containing serum and si-CMTM6 were applied to Huh7 cancer cells, and the proliferation, migration, and invasion abilities were evaluated by CCK-8 and Transwell assays, respectively. Protein expression levels of proliferating cell nuclear antigen (PCNA), epithelial-mesenchymal transition (EMT) markers, and CMTM6 were detected by Western blot. Results CMTM6 knockdown significantly reduced the mRNA and protein expression level of CMTM6 in Huh7 cells (P<0.05). There were no significant differences between the BC and NC groups in terms of cell proliferation, migration, invasion, expression levels of PCNA, EMT markers, and CMTM6 (all P>0.05). BJJP-containing serum markedly inhibited Huh7 cell proliferation, migration, and invasion (P<0.05), downregulated PCNA, CMTM6, N-cadherin, and Vimentin expression, and upregulated E-cadherin compared with the NC group (all P<0.05). CMTM6 knockdown suppressed malignant behaviors, with reduced PCNA, Vimentin, and N-cadherin and elevated E-cadherin expression (all P<0.05). Conclusions BJJP containing serum can significantly inhibit Huh7 cell growth, invasion, migration, and EMT progression, potentially mediated via CMTM6 suppression.