Objectives: Many studies have reported noticeable increases in the proportion of employees working either relatively short or relatively long hours. Such trends have been accompanied by an increasing concern that how much subjective mental well-being of employees would be influenced by their hours of work. The aim of this study was to investigate the association between work hours and clinically relevant depressive symptoms with demographic variables adjusted. Methods: Participants were employees of a total of 56 private companies and local government organizations in Korea, aged 19 to 65 years. A self-report questionnaire that included items on working hour, job stress, levels of depression, and socio-demographic factors was administered to 15360 Korean employees, with 14477 valid responses. Hierarchical linear regression analyses, adjusted for sociodemographic factors, job related demographic factors, job stress, were used additionally to estimate the association between working hours and depressive scores. Results: We found that working more than 40 hours per week correlated positively with the level of depressive symptoms after adjusting for demographic variables and the level of job stress. Furthermore, working 40 or fewer hours per week correlated negatively with the level of depressive symptoms. Being younger (β = -0.078, β = -0.099), being a female (β = 2.770, β = 1.268), and possessing a lower level of education (β = -0.315, β = -1.125) were significantly associated with higher level of depressive symptoms in all respondents. Conclusions Both of working excessively long or short hours is significantly associated with the prevalence of depressive symptoms. Establishing proper office hours for employees is critical to improving the quality of working conditions and maintaining good mental health in the workplace.