Despite a beneficial role of mushroom consumption against risk of type 2 diabetes, few studies investigated such associations in a prospective study. This study aimed to evaluate whether habitual mushroom consumption prospectively associate with risk of type 2 diabetes incidence among Korean men and women aged ≥ 40 years. A total of 16,666 participants of the cardiovascular disease association study (CAVAS) who did not have type 2 diabetes were included. Dietary mushroom was calculated using a food frequency questionnaire composed of 106 items. For 96,945 person-years of follow-up, 945 participants developed de novo type 2 diabetes. After adjustment for established diabetes risk factors and a modified Diet Quality Index-International (DQI-I), mushroom consumption was inversely associated with a risk of type 2 diabetes in both men and women (incidence rate ratios (IRR) for the highest vs. lowest quartile: IRR=0.65, 95% CI=0.47–0.90, P trend=0.0427 for men; IRR=0.70, 95% CI=0.54–0.93, P trend=0.1145 for women). These significantly associations remained robust when we adjusted for fasting blood glucose at baseline and excluding the first year of follow-up. In stratified analyses, the association between mushroom consumption and the risk of type 2 diabetes persisted in all subgroups, and no significant effect modification was observed between mushroom consumption and age, education level, regular exercise, smoking status, drinking status, obesity, pre-diabetes condition, or DQI-I score. Our findings indicate that a high dietary intake of mushroom is associated with lower risk of type 2 diabetes in both men and women.