Shift work have been thought to restrict participation in leisure time activities, but the knowledge about physical activity in rotating night shift nurses has been limited so far. We investigated the associations between the rotating night shift work and physical activity using data from a cross-sectional study among nurses and midwives. This study included 354 nurses and midwives (aged 40-60) currently working rotating night shifts and 371 ones working days only. The information on the work characteristics and potential covariates was collected via a personal interview. Weight and height were measured and BMI was calculated. Physical activity was assessed according to the international questionnaire on physical activity - IPAQ, and four domains: leisure time, occupational, transport related and household were analyzed. Women who reported none leisure time activity were defined as recreationally "inactive". The associations were examined with multiple linear or logistic regression models adjusted for age, season of the year, number of full term births, marital status and BMI. Total and occupational physical activity was significantly higher among nurses working rotating night shifts. However, leisure time activity was significantly affected among rotating night shift nurses and midwives, compared to women working during the days only, with increased odds ratio for recreational "inactivity" (OR = 1.57, 95% CI: 1.11-2.20). Rotating night shift work among nurses and midwives is associated with higher occupational physical activity but lower leisure time activity. Initiatives supporting exercising among night shift workers are recommended.