Background: Mediterranean diet adherence has been associated with reduced risks of various cancer types. However, prospective findings for overall cancer risk are inconclusive. Objective: The aim of this study was to examine sex-specific relations of Mediterranean diet adherence with overall cancer risk. Design: This analysis was conducted as part of the prospective Netherlands Cohort Study. Baseline data on diet and other cancer risk factors were collected using a self-administered questionnaire. Participants were followed up for cancer incidence for 20.3 years through record linkages with the Netherlands Cancer Registry and the Dutch Pathology Registry. The alternate Mediterranean diet score without alcohol was the principal measure of Mediterranean diet adherence. Participants/setting: The study population consisted of 120,852 inhabitants of the Netherlands, who were aged 55 to 69 years in September 1986. Main outcome measure: The primary outcome was overall cancer incidence. Statistical analyses performed: Cox regression analyses (case-cohort design) were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for associations of Mediterranean diet adherence with incidence of cancer (subgroups). In total, 12,184 male and 7,071 female subjects with cancer had complete data on potential confounders and were eligible for inclusion in the Cox models. Results: Middle compared with low Mediterranean diet adherence (alternate Mediterranean diet score without alcohol) was significantly associated with a reduced overall cancer risk in women (HR [95% CI]: 0.85 [0.75-0.97]). Decreased HR estimates for the highest Mediterranean diet adherence category and per 2-point increase in score were also observed, but did not reach statistical significance in multivariable-adjusted analyses. In men, there was no evidence of an association for overall cancer risk (HRper 2-point increment [95% CI]: 1.02 [0.95-1.10]). Results for cancer subgroups, defined by relations with tobacco smoking, obesity, and alcohol consumption, were largely similar to the overall findings. Model fits diminished when alcohol was included in the Mediterranean diet score. Conclusions: Mediterranean diet adherence was not associated with overall cancer risk in male participants of the prospective Netherlands Cohort Study. HR estimates in women pointed in the inverse direction, but lost statistical significance after full adjustment for confounding in most cases.