The biosynthesis and metabolism of RNA play important roles in regulating gene expression. On the other hand, it has been shown that RNA expression profiling is differentially distinct between cancer and normal cells, suggesting the possibility that aberrant regulation of RNA metabolism might be associated with the development and progression of cancer. In the current study, we found that Sam68, an RNA-binding protein that links cellular signalling to RNA processing, was markedly overexpressed in breast cancer cells and tissues. Immunohistochemical analysis showed that the expression and cytoplasmic localization of Sam68 significantly correlated with clinical characteristics of patients, including clinical stage, tumour-nodule-metastasis (TNM) classification, histological grade, and ER expression. Univariate and multivariate analyses showed that the expression level and cytoplasmic localization of Sam68 were identified as independent prognostic factors. Furthermore, we found that siRNA knockdown of endogenous Sam68 inhibited cell proliferation and tumourigenicity of breast cancer cells in vitro, through blocking the G1 to S phase transition. Moreover, we demonstrated that the anti-proliferative effect of silencing Sam68 on breast cancer cells was associated with up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) and p27(Kip1), enhanced transactivation of FOXO factors, and attenuation of Akt/GSK-3β signalling. Taken together, our results suggest that Sam68 might play an important role in promoting the proliferation and carcinogenesis of human breast cancer, and thereby might be a novel and useful prognostic marker and a potential target for human breast cancer treatment.