Objectives: The health benefits of breastfeeding are widely recognized. The World Health Organization recommends exclusive breastfeeding for six months after birth and for two years or longer together with nutritionally adequate complementary foods. To respond to the needs of industry, employed breastfeeding mothers must adapt to the rotating night shift (RNS). However, the RNS is associated with a higher risk of health problems in career women. We investigated the relationship between the RNS and breast milk volume. Methods: Mothers who used a breastfeeding room while working at a technology company in Taiwan voluntarily participated in this study from March 1 through April 30, 2013. We compared two groups: breastfeeding mothers on (RNS⁺) and not on a RNS (RNS⁻) to determine independent predictors for breast milk volume. We analyzed data from 109 participants: RNS⁺ group n=56; RNS⁻ group n=53. Results: There was no significant difference in daily milk collection volume between the groups. Daily milk collection frequency and exclusive breastfeeding were independent predictors for a daily breast milk collection volume >350 m l . Conclusions: The RNS may not affect the breast milk volume. This result may help the government and employers make policies more appropriate for supporting employed breastfeeding mothers.(J Occup Health 2015; 57: 81–86)