Double-strand breaks (DSBs), the most serious lesions of DNA, often induce chromosomal aberrations and are intimately associated with oncogenesis. A normal DNA damage response (DDR) network contains two major repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Studies of DSB repair-associated molecules have focused mainly on DNA repair proteins. However, non-coding RNAs also play important roles in the process of DSB repair. Over the past two decades, microRNAs (miRNAs) have been extensively investigated. Our previous work showed that miR-34c-5p overexpression results in suppression of RAD51 and upregulation of γH2AX. In accordance with this, we confirmed that miR-34s family overexpression increased endogenous DSB levels to different extents, an effect that was further confirmed to be associated with the decreased efficiency of HR repair. In addition, miR-34s overexpression also induced G1 arrest, inhibited proliferation and promoted apoptosis. As a central molecule in the process of HR pathway, RAD51 expression was strongly repressed in cells transfected with the miR-34a/b/c-5p mimic. Finally, we demonstrated that miR-34a/b/c-5p directly targets the RAD51 mRNA 3'-UTR or indirectly inhibits RAD51 expression via the p53 signaling pathway. Taken together, our results indicate that miR-34s overexpression depresses the efficiency of HR repair and induces DSBs by downregulating RAD51 expression. Our findings highlight a novel mechanism of HR pathway regulation via the miR-34s/p53/RAD51 axis.