Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of Janus kinase and the signal transducer and activation of transcription (Jak-STAT) pathway. SOCS-1 is known to be silenced by aberrant promoter methylation in human hepatocellular carcinoma (HCC) during early tumorigenesis, therefore, a strategy to restore SOCS1 expression can be utilized for cancer therapy. Here, we examined the influence of adenine nucleotide translocase 2 (ANT2) suppression by short-hairpin RNA (shRNA) on SOCS1 expression and its downstream effect in HCC. ANT2 shRNA treatment led to restoration of SOCS1 expression along with its promoter demethylation in Hep3B cells, which was accompanied by decreased DNA methyltransferase 1 (DNMT1) activity through the suppression of Ras/PI3K/Akt signaling. Restoration of SOCS1 by ANT2 knockdown, subsequently, inhibited STAT3 activity and downregulated the expression of miR-21, which has been reported to be an important onco-miR in HCC. Downregulation of miR-21 efficiently suppressed Hep3B cell proliferation in vitro with a comparable level to ANT2 shRNA treatment. ANT2 suppression by shRNA may be able to exert anticancer effects in HCC further by restoring SOCS1 expression.