Several case-control studies have reported that mushroom consumption may be associated with reduced risk of certain cancers. However, epidemiologic studies have not yet prospectively examined the association of mushroom consumption with total and various site-specific cancer risk. This prospective cohort study included 68,327 women (Nurses' Health Study, 1986-2012) and 44,664 men (Health Professionals Follow-up Study, 1986-2012) who were free of cancer at baseline. Mushroom consumption was assessed at baseline using a validated food frequency questionnaire. Covariates were assessed using biennial questionnaires during the follow-up. We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of total and 17 site-specific cancers associated with mushroom consumption. During up to 26 years of follow up, we documented 22,469 incident cancer cases (15,103 in women and 7,366 in men). In the pooled multivariable analysis, participants who consumed 5 or more servings of mushrooms per week had no significantly different risk of total cancer (HR=1.06, 95% CI=0.98-1.14) than participants who almost never consumed mushrooms. We consistently found no association between mushroom consumption and risk of 16 site-specific cancers. However, there was a marginal positive association between mushroom consumption and risk of lung cancer (P-trend=0.05). In conclusion, we found no association between mushroom consumption and total and site-specific cancers in US women and men. More prospective cohort studies are needed to examine the associations for specific cancer types in diverse racial/ethnic groups.