Deleted in liver cancer 2 (DLC2) is a tumor suppressor, associated with various types of cancer. The aim of the present study was to analyze the expression of DLC2 in breast cancer, its clinical significance and its effect on breast cancer cell behavior. The expression of DLC2 was evaluated by immunohistochemistry in 131 cases of breast cancer. Associations among DLC2 expression and clinicopathological features were analyzed, and its effects on proliferation, motility, migration and invasion in DLC2-knockdown breast cancer cell lines were observed. The results indicated that DLC2 was expressed in 42.75% of breast cancer cases (56/131) and in 79.39% of adjacent normal tissues (104/131). Lower expression of DLC2 in breast cancer was associated with tumor differentiation (P<0.001), lymph node metastasis (P<0.001) and poor prognosis (P<0.001). The silencing of the DLC2 gene in human breast cancer cell indicated an increased number of cells entering S phase, and increased abilities of clone formation, cell migration and invasion. Downregulated expression of DLC2 was associated with activated Ras homolog family member A and decreased Rac family small GTPase 1, cell division cycle 42 and Rho-associated protein kinase-2 expression levels, indicating that DLC2 may serve a regulatory function in breast cancer cell proliferation and invasion via the RhoGTPase pathway. The results of the present study suggested that DLC2 serves as a suppressor gene in the development of breast cancer and may be a prognostic marker for patients with breast cancer.