The cancer‐testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progress of hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, the RNA interfering (RNAi) method was used to silence CT23 expression in BEL‐7404 cells. Microarray analysis was performed on mRNA extracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two‐fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformatic analysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attained the maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non‐neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL‐7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight into searching for a novel therapeutic target for HCC.