SThis research aimed to explore effects of SIX1 and DACH1 on hepatocellular carcinoma (HCC) cell proliferation, apoptosis and cell cycle. Fifty paired hepatocellular carcinoma tissues were screened for differentially expressed genes. SIX1 and DACH1 expressions were subjected to qRT-PCR and western blot in tumor tissues and cells. The knockdown efficiency of siRNAs and transfection efficiency of cDNAs and siRNAs were validated by qRT-PCR and western blot as well. Then colony formation assay and flow cytometry were applied to observe cell proliferation, cell apoptosis and cell cycle changes. Immunofluorescence co-localization and immunoprecipitation were used to analyze the interaction between proteins which was quantified using western blot. Effects of SIX1 and DACH1 on tumor growth and their expressions in tumors were confirmed in vitro in nude mice model. Results of these experiments showed that SIX1 was overexpressed while DACH1 was suppressed in HCC tissues and cells. The suppression of SIX1 and overexpression of DACH1 not only inhibited cell proliferation, but also induced cell apoptosis and arrested cell cycle in G2/M phase compared with control group. Results of immunofluorescence co-localization suggested that SIX1, p53 and DACH1 were significantly overlapped. Immunoprecipitation showed that DACH1 (marked with Flag tag) could pull down p53 and SIX1, but SIX1 (marked with His tag) could only pull down DACH1, which indicated that an indirect regulation between SIX1 and p53. Validated with western blot afterwards, DACH1 overexpression suppressed tumorigenesis in vivo by up-regulating p53 expression while SIX1 overexpression accelerated tumor growth by down-regulating p53 expression. Therefore, the decrease of SIX1 facilitated the expression of DACH1, thus activated the expression of p53 and suppressed the progression of HCC both in vitro and in vivo.