The modern 24/7 society demands night shift work, which is a possible risk factor for chronic diseases. This study aimed to examine the associations of rotating night shift work duration with tooth loss and severe periodontitis. This cross-sectional study used data from a self-administered questionnaire survey conducted among 3,044 permanent employees aged 20-64 years through a Japanese web research company in 2023. The duration of rotating night shift work was assessed using a question from the Nurses' Health Study. Tooth loss was assessed based on self-reported remaining natural teeth count. Severe periodontitis was assessed using a validated screening questionnaire comprising four questions related to gum disease, loose tooth, bone loss, and bleeding gums. We employed linear regression models for tooth loss and Poisson regression models for severe periodontitis, adjusting for demographic, health and work-related variables and socioeconomic status. Among participants included, 10.9% worked in rotating night shifts for 1-5 years, while 11.0% worked in such shifts for ≥6 years. In fully adjusted models, rotating night shift work duration of 1-5 years was associated with tooth loss (beta -0.74, 95% confidence interval (CI) [-1.55 to 0.08]) and severe periodontitis (prevalence ratio 1.80, 95% CI [1.33-2.43]); however, the association with tooth loss was not statistically significant. This study supports that employees who work short-term rotating night shifts may experience poor oral conditions. Further research is needed to determine whether long-term rotating night shift work is associated with deteriorated oral health.