Nod-like receptor pyrin domain-containing protein 6 (NLRP6) plays a key role in innate immunity, host defense and tumorigenesis. Our previous study has demonstrated the tumor suppressor role of NLRP6 in gastric cancer. In the present study, we explored the interaction protein of NLRP6 by Flag-tagged immunoprecipitation assay and liquid chromatography/mass spectrometry-based proteomics analysis. The 78 kDa glucose-regulated protein (GRP78), a heat shock protein, was identified as an interaction protein of NLRP6. The binding of NLRP6 to GRP78 was through the Pyrin domain, and the substrate binding domain (SBD) domain of GRP78 was responsible for the interaction with NLRP6. NLRP6 overexpression enhanced the polyubiquitination of GRP78 in gastric cancer cells. Overexpression of GRP78 abolished the effects of NLRP6 overexpression in gastric cancer cell proliferation, cell cycle progression, cell apoptosis, migration and Cyclin D1 expression. GRP78 knockdown reversed the effects of NLRP6 knockdown on cell proliferation and cell cycle progression. NLRP6 expression was negatively correlated with GRP78 expression in human gastric tissues. Tumorigenicity assay indicated that GRP78 mediated the functions of NLRP6 on gastric cancer cell growth in vivo. ON-013100, which could inhibit Cyclin D1 expression, was less effective in treating xenografts of gastric cancer cells with higher level of NLRP6 than in those with lower level of NLRP6. In conclusion, our study suggested that NLRP6 exerted inhibitory effects on gastric cancer cell growth by promoting the ubiquitination of GRP78.