Many key proteins are down-regulated or lose their function during cancer genesis and accelerate the progress of cancer. We found that nuclear apoptosis-inducing factor 1 (NAIF1) was highly expressed in normal gastric tissues but was down-regulated or lost in gastric cancer tissues (P<0.001). NAIF1 expression was higher in well-differentiated (P=0.004) than in moderately- or poorly-differentiated gastric cancer. NAIF1 expression was associated with different T stages (P=0.024). In vitro, NAIF1 can inhibit tumor cell proliferation and induce G0/G1 phase cell cycle arrest in the MKN45 cell line. NAIF1 can induce apoptosis through activation of procaspase-9 rather than procaspase-8 followed by activation of the caspase-3 pathway. We designed and constructed two truncation mutants, pEGFP-N1-NLS and pEGFP-N1-GRR, and identified the N-terminal 1-90 amino acid domain of NAIF1, which is a helix-turn-helix motif and which was sufficient for inducing apoptosis. Therefore, these findings suggest that NAIF1 plays an inhibitory role in the initial steps of gastric cancer genesis and may provide new strategies for developing anti-cancer drugs using small molecular polypeptides.