Introduction: Sedentary behavior (including prolonged sitting) is a form of physical inactivity that has a negative impact on health, possibly including musculoskeletal complaints (MSCs). The purpose of this study was to determine the extent to which time spent sitting at work is associated with the one-year prevalence of MSCs in the neck, shoulder, upper back/thoracic spine, and lower back among workers from the Study of Mental Health in the Workplace (S-MGA). In addition, the study also examined whether leisure time, physical activity, and sex modify the relationship between occupational sitting and MSCs. Methods: For this analysis, we used the S-MGA, a 5-year prospective study in Germany. The S-MGA is a nationwide representative employee cohort study with a baseline survey in 2012 and a follow-up survey in 2017. Sitting at work was measured using a question asked at baseline. The Nordic Musculoskeletal Questionnaire was used to determine the one-year prevalence of MSCs in the neck, shoulder, upper back, and lower back pain (yes/no). The assessment of MSCs was only conducted at the 2017 follow-up. Adjusted Poisson regression models were used to determine the association of baseline level of weekly hours spent sitting at work with MSCs during follow-up. In addition to unadjusted models, models were adjusted for demographic (age, sex, body mass index and occupational skill level), occupational (heavy lifting at work), psychological disorders and lifestyle factors (smoking status and leisure time physical activity), as well as preexisting musculoskeletal conditions reported at baseline. To examine whether the relationship between sitting time and pain was modified by sex and leisure time physical activity, the models were stratified for both these variables. Results: Among the participants analyzed (n = 2,082), 49.8% were male, while 50.2% were female, and more than 60% of the study population spent over half of their working hours in a sitting position. Exposure to increased sitting at work reported at baseline was not consistently associated with 12-month prevalence of MSCs in the upper body at follow-up. However, differences in the association between occupational sitting and MSCs were dependent on the intensity of leisure time physical activity. Prevalence ratios (PRs) indicated an increased prevalence of MSC in the neck (PR = 1.46; 95% CI = 1.18–1.80) and shoulder (PR = 1.30; 95% CI = 1.03–1.64) in workers without leisure time physical activity who spent 25 to < 35 weekly working hours sitting. Discussion: These findings suggest that leisure time physical activity interacts with the relationship between sitting at work and MSCs. The relationship between sitting at work and musculoskeletal pain needs further investigation, but we found indications that leisure time physical activity may counter the effects of sitting at work.