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OBJECTIVES: The aim of this study was to investigate whether melatonin receptor type 1B (MTNR1B) rs10830963 polymorphism interacts with night shift work on the risk of incident stroke. METHODS: This study included individuals free of stroke at baseline from the UK Biobank. Night-shift work was assessed by the self-reported questions. MTNR1B rs10830963 was directly genotyped (CC, GC, and GG). Incident stroke was ascertained through hospital records and death registries. Cox proportional hazards models were employed to examine the associations of night shift work and MTNR1B rs10830963 with the risk of incident stroke. RESULTS: A total of 242 194 participants were finally included (mean age: 52.95 years; 51.63% women). Over 12-year follow-up, 3287 incident stroke events occurred. Night shift work increased the risk of incident stroke [hazard ratio (HR) 1.13, 95% confidence interval (CI) 1.00–1.28] after adjusting for socio-demographics, and this association attenuated after additional adjustment for lifestyle factors (HR 1.06, 95% CI 0.94–1.20). MTNR1B rs10830963 polymorphism modified the association between night shift work and incident stroke (Pfor interaction =0.010). In the Cox models adjusted for socio-demographics and lifestyle factors, among night-shift workers, minor allele G was associated with a reduced risk of incident stroke (GC versus CC, HR 0.74, 95% CI 0.58–0.95; GG versus CC, HR 0.65, 95% CI 0.40–1.06; P for trend=0.010); while night shift work was associated with a higher stroke risk only among MTNR1B rs10830963 CC carriers (HR 1.23, 95% CI 1.05–1.44) but not GC/GG carriers. CONCLUSIONS: These results suggest that MTNR1B rs10830963 may potentially modify the associations between night shift work and incident stroke.