The regular practice of physical activity (PA) has been associated with better health. In addition, job characteristics may determine the PA behaviours of employees, including the work shift. However, relatively few studies have examined the PA behaviour among shift workers.This study aimed to investigate the association between work shift and the practice of PA among workers of a poultry processing plant in Southern Brazil.A cross-sectional study was conducted with 1206 workers (786 females), ages 18 to 50, working in shifts on a production line that operates 24 hours/day. Workers who engaged in more than 150 minutes/week of PA were considered active. Multivariate analyses were conducted using Poisson regression and all analyses were stratified by gender.Of the total participants studied, 36% (95%CI: 33-39) were considered active and the sociodemographic characteristics associated with PA differed among males and females. Regarding work shift, night shift workers had higher prevalence of PA. However, increased PA was significantly associated with work shift particularly among females. After controlling for potential confounders, women who worked during the night shift were approximately 30% more active than those who worked during the day (PR[prevalence ratio]= 1.32;95%CI: 1.07-1.62;p=0.010).This study found a low prevalence of PA among the workers and indicated a significantly association between work shift and PA. Working at night was positively associated with regular PA, particularly among females. These results contribute for initiatives that aim to increase PA among the workers, take into account the job characteristics and gender differences.