Objective This study aimed to investigate a prospective association between shift work and use of psychotropic medicine. Methods Survey data from random samples of the general working population of Denmark (N=19 259) were linked to data from national registers. Poisson regression was used for analyses of prospective associations between shift work and redeemed prescriptions of psychotropic medicine. Prevalent cases were excluded at baseline. In secondary analyses, we tested differential effects on subsets of psychotropic medicine and, cross-sectionally, we studied correspondence between estimates based on psychotropic medicine and self-reported mental health. According to the protocol we interpret results from the secondary analyses following the principles for nested hypothesis testing, if the primary analyses reject the null-hypothesis, and otherwise we regard it as hypothesis generating exploratory analyses. Results In the primary analysis, the rate ratio for incidence of psychotropic medicine among shift workers was 1.09 (95% confidence interval 0.99–1.21). Results from the secondary analyses suggested increased incidence of use of hypnotics, sedatives and antidepressants and decreased incidence of use of anxiolytics. Cross-sectional analysis suggested increased risk for use of psychotropic medicine (all kinds), but not for poor self-rated mental health. Conclusions Results did not support that working in shifts to the extent that is currently practiced in Denmark is associated with an increased incidence of overall psychotropic medicine use. Future studies should test, whether there is a differential incidence for different drugs among shift workers as suggested by the secondary analyses and how psychotropic medicine use and mental health are related.