OBJECTIVE: MLN4924 is a second-generation small molecule inhibitor with anti-cancer activity that inhibits neddylation activation enzyme (NAE), subsequently blocking the neddylation-dependent activation of Cullin-RING E3 ligases (CRLs). Mof4 family associated protein 1 (MRFAP1) is a highly conserved, short half-life protein and one of the most up-regulated proteins in response to MLN4924 treatment. MRFAP1 has been identified as a novel cell cycle-related protein and a regulatory component monitoring and preventing genomic instability. However, whether MRFAP1 plays a role in MLN4924-mediated cancer cell death remains elusive. PATIENTS AND METHODS: The expression of MRFAP1 in gastric cancer clinic samples was detected by Real-time PCR and Western blot. CRIS-PR (clustered regularly interspaced short palindromic repeats)/Cas9 system was used to knock-out MRFAP1 gene in both AGS and SGC-7901 cells. The proliferation of GC cells was measured by CCK8 assay. The cell cycle distribution of GC cells was determined by fluorescence-activated cell sorting (FACS) assay. Co-immunoprecipitation assay was used to determine the interaction between MRFAP1 and P27. RESULTS: MRFAP1 was downregulated in clinic gastric cancer samples at post-translational level. Overexpression of MRFAP1 decreased gastric cancer cells proliferation. CRISPR-mediated knockout of MRFAP1 increased the cytotoxicity of MLN4924 by augmenting MLN4924-induced G2/M arrest and apoptosis against gastric cancer cells. At the molecular level, we found that MLN4924 induced the interaction between P27 and MRFAP1, the latter associated with P27, which was further stabilized in response to MLN4924 treatment. CONCLUSIONS: We showed a protective role of MRFAP1 in gastric cancer cells with MLN4924 treatment and suggested the potential possibility to combine MLN4924 with MRFAP1 inhibition to treat gastric cancer.