n-3 polyunsaturated fatty acids (PUFAs) and n-6 PUFAs are reported to have immunomodulatory effects, but few studies have examined these functions. Thus, we examined whether dietary n-3 and n-6 PUFAs are associated with the risk of gastric cancer and further investigated whether fatty acid desaturases 1 and 2 (FADS1 and FADS2) modify this association. In a case-control study, 1,464 participants (402 cases and 1,062 controls) were enrolled. A semi-quantitative food frequency questionnaire was utilized to measure dietary PUFA intake. Genotyping was performed using the Axiom® Exome 319 Array. Multivariable logistic models were established after adjusting for confounding variables. The risk of gastric cancer was significantly decreased among participants who had the highest tertile intake of docosahexaenoic acid (DHA), an n-3 PUFA, even after adjusting for covariates [odds ratios (OR) = 0.72, 95% confidence intervals (95% CIs) = 0.53-0.99]. However, no significant interaction according to FADS1 rs174546 or FADS2 rs174583 was observed. In conclusion, we observed a significant inverse association between dietary DHA and the risk of gastric cancer but found that FADS1 rs174546 and FADS2 rs174583 did not modify the association between dietary n-3 or n-6 PUFAs and gastric cancer risk.