Objective: This cross-sectional multilevel study aimed to investigate the relationship between improvement of the work environment and work-related stress in a nationally representative sample in Japan. Methods: The study was based on a national survey that randomly sampled 1745 worksites and 17,500 nested employees. The survey asked the worksites whether improvements of the work environment were conducted; and it asked the employees to report the number of work-related stresses they experienced. Multilevel multinominal logistic and linear regression analyses were conducted. Results: Improvement of the work environment was not significantly associated with any level of work-related stress. Among men, it was significantly and negatively associated with the severe level of work-related stress. The association was not significant among women. Conclusions: Improvements to work environments may be associated with reduced work-related stress among men nationwide in Japan.