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Abstract Aims/Introduction The role of irisin in maternal glucose metabolism and how it would respond to dietary n‐3 polyunsaturated fatty acid (n‐3 PUFA) intake remains unclear. This study aimed to explore whether maternal plasma irisin is associated with glucose metabolism and whether this association is modified by dietary n‐3 PUFA. Materials and Methods A total of 932 pregnant women (20–28 weeks’ gestation) aged 20–45 years were recruited. Dietary n‐3 PUFA was estimated using a validated quantitative food frequency questionnaire. Plasma irisin and insulin were tested by enzyme‐linked immunosorbent assay, and insulin resistance (IR) was estimated using the homeostatic model assessment (HOMA). Gestational diabetes mellitus was diagnosed with a 75‐g oral glucose tolerance test. Adjusted multivariable linear regression and logistic regression were carried out to examine the associations between plasma irisin and glucose metabolism. The moderating effect of dietary n‐3 PUFA intake was determined by fully multiplicative models by including the interaction term. Results Maternal plasma irisin was negatively associated with HOMA‐IR and oral glucose tolerance test 0 h glucose level (β −0.250, −0.067; corrected P‐value for false discovery rate = 0.012, 0.018, respectively), positively associated with HOMA of insulin sensitivity (β 0.028; corrected P‐value for false discovery rate = 0.012), but not associated with postprandial glucose or the risk of gestational diabetes mellitus. Furthermore, we found a moderating effect of dietary n‐3 PUFA on the relationships of plasma irisin with HOMA‐IR and HOMA of insulin sensitivity; these associations were strengthened with increased n‐3 PUFA intake (β −0.037, 0.004; P = 0.014, 0.041, respectively). Conclusions Plasma irisin was negatively associated with HOMA‐IR and fasting glucose, whereas it was positively associated with HOMA of insulin sensitivity in pregnant women. We first showed that these associations were modified by dietary n‐3 PUFA intake.