Abstract Background: Suppression of tumorigenicity 5 (ST5) has been considered as a tumor suppressor gene in HeLa tumor cells. However, there is no report of ST5 expression or function in the progression of breast cancer.Methods: ST5 expression in different subtypes and pathological stages of breast cancer was determined by Oncomine database, Breast Cancer Gene-Expression Miner v4.4 (bc-GenExMiner v4.4) analysis and immunohistochemistry. Cell viability was measured by CCK8 assay and metastatic behavior was assessed using scratch wound model and Transwell. Flow cytometry was employed for cell cycle and apoptosis detection, and methylation-specific PCR (MSP) was used to detect methylation level.Results: ST5 was expressed at low level in different subtypes of breast cancer specimens compared to normal breast and there was a negative association between ST5 status and pathological stages of breast cancer patients. Additionally, ST5 was lower in cases of recurrent and invasive breast cancer than that in non-recurrent and non-invasive patients. In in vitro experiment, ST5 status was also negatively associated with the invasive capability of breast cancer cells, showing lower in MDA-MB-231 and SKBR3 cell lines than that in MCF-7 cells. ST5-downregulation promoted, while ST5-upregulation inhibited the tumour characteristics of MDA-MB-231 cells including cell viability, cell cycle and migration. And exogenous ST5 also elevated, but ST5 depletion limited the proportion of apoptotic cells in MDA-MB-231 cells. However, the alteration of ST5, no matter upregulation or downregulation, had no impact on tumour behaviors of MCF-7 cells. Mechanistically, ST5 protein ablation activated, while ST5-upregulation repressed the activities of phosphorylated JNK and ERK1/2, and subsequently the expression of c-Myc. Of note, low level of ST5 in breast cancer cells was possibly related with the aberrant methylation of ST5 promoter region.Conclusion: Our findings suggest that ST5 potentially acts as a tumor suppressor gene in invasive breast cancer through regulating ERK/JNK signaling pathway and provide a novel insight for breast cancer treatment.