Abstract Background Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. The only drug currently approved for clinical use in the treatment of advanced HCC is sorafenib. However, many patients with HCC show reduced sensitivity to sorafenib during treatment. SIRT3, a member of the mammalian sirtuin family, is a tumor suppressor in certain tumor types. However, only few studies have investigated the effects of SIRT3 on tumor prognosis and sorafenib sensitivity in patients with HCC. Here, we aimed to investigate the correlation between SIRT3 expression and glucose metabolism and proliferation in HCC and discover effective compounds that increase endogenous SIRT3 modulation effect of sorafenib. Methods To determine the correlation between SIRT3 and glucose related proteins, immunostaining was performed with liver cancer tissue using various antibodies. To investigate whether the expression of SIRT3 in HCC is related to the resistance to sorafenib, we treated sorafenib after the modulation of SIRT3 levels in HCC cell lines (overexpression in Huh7, knockdown in HepG2). We also employed PD0332991 to modulate the SIRT3 expression in HCC cell and conducted functional assays. Results SIRT3 expression was downregulated in high glycolytic and proliferative HCC cells of human patients, xenograft model and HCC cell lines. Moreover, SIRT3 expression was downregulated after sorafenib treatment, resulting in reduced drug sensitivity in HCC cell lines. To enhance the anti-tumor effect of sorafenib, we employed PD0332991 (CDK4/6-Rb inhibitor) based on the correlation between SIRT3 and phosphorylated retinoblastoma protein in HCC. Notably, combined treatment with sorafenib and PD0332991 showed an enhancement of the anti-tumor effect in HCC cells. Conclusions Our data suggest that the modulation of SIRT3 by CDK4/6 inhibition might be useful for HCC therapy together with sorafenib, which, unfortunately, has limited efficacy and whose use is often associated with drug resistance.