Objective To explore the biological effect of transcription factor B1, mitochondrial (TFB1M) on hepatocellular carcinoma (HCC) cells, and to explore the molecular mechanism of its regulation on malignant metastasis of HCC. Methods The expression level of TFB1M in liver cancer and its relationship with clinical characteristics of patients with HCC was analyzed based on TCGA-LIHC and GEO databases. Based on the median expression level of TFB1M, the TCGA-LIHC samples were divided into low expression group of TFB1M (n=170) and high expression group of TFB1M (n=170), and GSEA was used for gene set enrichment analysis.The overexpressed TFB1M plasmid was constructed to transfect HepG2 and Huh7 cells. Cell migration and invasion were assessed by scratch healing and Transwell chamber assays. Results The expression of TFB1M was significantly decreased in HCC, and the expression of TFB1M was related with gender in HCC. The Results of GSEA analysis indicated that the low expression of TFB1M might be related to liver cancer subclass proliferation up gene set, stem cell hepatocellular carcinoma gene set, and extracellular matrix assembly gene set in HCC. Overexpression of TFB1M remarkably attenuated the migration and invasion activity of HCC cells. Conclusions The expression of TFB1M was significantly decreased in HCC, and overexpression of TFB1M could attenuated the migration and invasion activity of HCC cells.