Colorectal cancer (CRC) is a typical and lethal digestive system malignancy. In this study, we investigated the effect of Sirtuin 3 (SIRT3) expression, a fidelity mitochondrial protein, on proliferation of CRC cells and the related mechanisms. Using UALCAN database and CPTAC database, we found low expression of SIRT3 in CRC was the unfavorable factor for survival prognosis. Meanwhile，SIRT3 expression was correlated with distant metastasis and TNM stage of CRC patients. Subsequently, we found the proliferating capacities of CRC cells with stably expressed SIRT3 were decreased dramatically in vitro and in vivo, with comparison to their counterparts, respectively. Further western blot (WB), immunoprecipitation (IP) and TOPflash/FOPflash assay showed the related mechanism of growth retardation of these cells was highly associated with the degradation of β-catenin in cytosol, and the localization of β-catenin/α-catenin complex in nucleus. Taken together, these results revealed the retardation of CRC cell proliferation by SIRT3 was highly correlated with the inactivation of Wnt/β-catenin signaling.