This cross-sectional study analyzed the association between physical activity (PA) and social jetlag (SJL) among shift workers. We evaluated 423 employees, aged between 18 and 60 years, who worked in a poultry-processing company in Brazil. Physical activity levels (PAL) were determined by the short form of the International Physical Activity Questionnaire (IPAQ-SF). SJL was calculated as the absolute difference between the time of mid-sleep on work and free days. The percentage of workers insufficiently active was high on all shifts, with no significant differences between the 3 shifts. The amount of time spent in sedentary behavior on workdays did not differ between shifts. Among night-shift workers, negative associations were observed between SJL and 1) the duration and weekly energy expenditure while walking; 2) the frequency of moderate and vigorous PA; and 3) the total energy expenditure related to PA. Even though PAL did not differ among workers on different shifts, we found a high prevalence of subjects who were insufficiently active or too sedentary among all groups. Also, SJL was negatively associated with PA in night-shift workers. These results suggest that PA should be encouraged among shift workers and that SJL may be an important factor in reducing PA in this cohort.