Gastric cancer (GC) seriously impacts on human health with high incidence and mortality rate in the world. Raf kinase inhibitor protein (RKIP) that was discovered in our previous studies is down-regulated in GC, which is associated with the occurrence, differentiation, invasion, and metastasis of GC. In order to investigate the molecular mechanisms and biological functions of RKIP in the occurrence and metastasis of GC, the fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transiently transfected into SGC7901 cells with liposome-mediated approach, the RKIP fusion proteins were purified with 3xFLAG tag affinity chromatography, and the RKIP-interacted proteins were identified with electrospray ionization-Quadrupole-time of flight (ESI-Q-TOF) tandem mass spectrometry (MS/MS). A total of 66 RKIP-interacted proteins were MS/MS-identified. The MS/MS-characterized components of the existing interaction complex (RKIP and 14-3-3) were confirmed with Western blotting in combination with co-immunoprecipitation. It is the first time to discover the interaction of RKIP with 14-3-3. This preliminary study on the molecular mechanisms that how RKIP inhibits the occurrence and development of GC provides novel clues for the early-stage diagnosis, prognosis monitoring, and targeted gene therapy of GC.