Breast cancer is the most common type of cancer and the most frequent cause of cancer deaths among women worldwide. MicroRNAs (miRNAs) are often dysregulated in various types of human cancer and can function as tumour suppressors or oncogenes in tumourigenesis. MiRNA-331-3p (miR-331-3p) plays an important role in hepatocellular carcinoma, colorectal cancer and glioblastoma; however, its expression level, roles and underlying mechanism in breast cancer remain unknown. Thus, this study investigated the expression level, biological roles and underlying mechanism of miR-331-3p in breast cancer. Results showed that miR-331-3p was significantly up-regulated in breast cancer tissues and cell lines. Down-regulation of miR-331-3p expression inhibited the proliferation and invasion of breast cancer cells. Src kinase signalling inhibitor 1 was validated as a novel direct target of miR-331-3p. Our findings revealed that miR-331-3p might serve as an oncogene, and its over-expression in tumour tissues might contribute to the carcinogenesis and progression of breast cancer. Thus, miR-331-3p is potentially a new therapeutic target for the treatment of breast cancer.