Mammary serine protease inhibitor B5 (SerpinB5) is a potential oncogene in gastric cancer (GC); however, the molecular mechanism by which SerpinB5 promotes oncogenesis remains elusive. In this study, SerpinB5-associated proteins were selected based on yeast two-hybrid screening and microarray analysis after RNA interference and were validated using co-immunoprecipitation (Co-IP) and RNA Co-IP. The expression profiles of the interacting proteins were analyzed by Western blotting and immunohistochemistry. The effects of SerpinB5 on KHDRBS3 and FBXO32 expression in GC cells were analyzed using real-time PCR and Western blotting after the expression of SerpinB5 was modified. By yeast two-hybrid screening and microarray analysis, FBXO32 and KHDRBS3 were found to be SerpinB5-interacting proteins. The interactions were confirmed by Co-IP. An RNA co-immunoprecipitation assay found that KHDRBS3 interacted with FBXO32 mRNA. The expression of SerpinB5 was much stronger in the nucleus of GC cells. FBXO32 was expressed at higher levels in the cytoplasm of GC cells. KHDRBS3 was primarily detected in the nucleus of normal mucosal cells. SerpinB5 expression was modified in GC cells, KHDRBS3 mRNA levels remained stable, however, FBXO32 mRNA levels changed 24 h after changes in KHDRBS3 protein levels were detected. In conclusion, SerpinB5 interacts with KHDRBS3 and FBXO32, and KHDRBS3 can interact with FBXO32 mRNA.