[Objective] To investigate the effect of CYB5D2 on tumor growth in s.c. transplantation mouse model of human breast cancer. [Method] Human breast cancer cells MCF-7 cells were transplanted s.c. to mice to generate tumors. Rats receiving multi-point injection of MCF-7 cells containing CYB5D2 were set as observation group; rats receiving multi-point injection of MCF-7 cells containing shRNA-CYB5D2 were set as neg. control group, and those receiving PBS injection were set as blank control group. Tumor volume and mass were measured. After model establishment, qRT-CPR was used to detect the mRNA expression level of CYB5D2 in tumor tissues, Western Blot was used to detect the expression of epithelial-mesenchymal transition (EMT) related markers in metastatic tumor tissues, and meantime, ELISA was used to detect the expression of MMP-2 and MMP-9 in the serum of metastatic tumor cells in nude mice. [Result] The average volume and mass of metastatic tumor in observation group were significantly lower than those in neg. control group and blank control group (P<0.05), and no significant difference was found between neg. control group and blank control group (P>0.05). The qRT-CPR method showed that the mRNA expression of CYB5D2 in tumor tissues of observation group was significantly higher than that of neg. control group and blank control group (P<0.05), and the expression showed no significant difference between neg. control group and blank control group (P>0.05). Western Blot showed that the expression levels of E-cadherin and β-catenin protein in metastatic tumor tissues of observation group were significantly higher than those of neg. control group and blank control group (P<0.05), and the expression levels of N-cadherin and Snail protein were significantly lower than those of neg. control group and blank control group (P<0.05). No statistical difference was found in the expression of E-cadherin, β-catenin, N-cadherin and Snail protein between neg. control group and blank control group (P>0.05). Compared with neg. control group and blank control group, observation group had significantly lower levels of MMP-2 and MMP-9 in serum (P<0.05), and the levels of the rest groups showed no statistical difference (P>0.05). [Conclusion] Up-regulation of CYB5D2 gene can effectively inhibit tumor growth in s.c. transplantation mouse model of human breast cancer, and its anti-tumor effect may be related to the down-regulation of N-cadherin, Snail, MMP2 and MMP9, as well as the up-regulation of E-cadherin and β-catenin, and the inhibition of EMT.