The E3 ligase RING finger and WD repeat domain 3 (RFWD3) can stabilize p53 in response to DNA damage, participate in replication checkpoint, and have an important role in multiple myeloma, testicular germ cell tumor and lung carcinogenesis. Its expression and molecular mechanisms have never been explored in gastric cancer. In present study, the RFWD3 was over-expressed in both AGS and HGC-27 gastric cancer cells are found. Knockdown of RFWD3 suppressed cells proliferation activity of gastric cancer cells. And further study showed down-regulation of RFWD3 promotes cell apoptosis, suppresses cell migration and invasion and blocks G2/M cell cycle progression, which may be related with AKT, ERK/P38 and Slug pathways. Collectively, the results of the present study showed that RFWD3 might be a candidate oncogene for gastric cell proliferation and may have an important role in gastric carcinogenesis.