Ochratoxin A (OTA), a mycotoxin produced by ubiquitous Aspergilli, is carcinogenic, teratogenic, and nephrotoxic in both humans and animals. Our previous study found that OTA induced DNA double-strand breaks (DSBs) and resulted in G2 phase arrest in human gastric epithelium immortalized (GES-1) cells. DSBs can cause genomic instability, mutations, and neoplastic transformations, and improper repair of DSBs may lead to the development of cancer. Rad51 is a key protein in the homologous recombination (HR) pathway of DSBs repair. The roles of Rad51 in the repair of DNA damage vary in response to different types of cytotoxic agents. The effect of OTA on Rad51 expression and its putative role in the OTA-induced DSBs in GES-1 cells are still not clear enough. The aim of the current study is to elucidate the role of Rad51 in OTA-induced DSBs in GES-1 cells. The results showed that OTA treatment decreased Rad51 expression in a dose- and time-dependent manner. Specific downregulation of Rad51 by siRNA induced DSBs and G2 phase arrest. Rad51 overexpression by transfection with a Rad51-expressing plasmid partly rescued the DSBs and G2 phase arrest in OTA-treated cells. The findings indicate that downregulation of Rad51 contributes to OTA-induced DNA damage in GES-1 cells. Knockdown of p53 with siRNA for 48h effectively reversed the downregulation of Rad51, and decreased the OTA-induced DSBs in GES-1 cells. In addition, the downregulation of Rad51 induced by OTA could be significantly attenuated with specific ERK inhibitor PD98059 or specific p38 MAPK inhibitor SB203580 pre-treatment in GES-1 cells. Thus, the results suggest that downregulation of Rad51 participates in OTA-induced DNA double-strand breaks in GES-1 cells in vitro. And p53, ERK and p38 signaling pathways are all involved in the process.