This study examined the association between long working hours and psychosocial stress responses. In total, 1,122 white-collar workers from a company in Korea completed self-administered questionnaires following a lecture about the study aim, procedures, and confidentiality. Psychosocial stress responses were evaluated using the Psychosocial Well-being Index - Short Form (PWI-SF), and psychosocial working conditions were evaluated with the Korean Occupational Stress Scale - Short Form (KOSS-SF). Multivariate logistic regression analysis was performed after adjusting for demographic variables and psychosocial working conditions to examine associations between long working hours and psychosocial stress responses. In comparison with the reference group, which worked 40–44 hours per week, the crude odds ratio (OR) of the respondents who worked 60 or more hours was 4.56 (95% confidence interval (CI), 2.55–8.15) in terms of psychosocial stress responses. After adjusting for demographic variables, the adjusted OR of those working ≥60 hours was 5.61 (95% CI, 3.01–10.47). After adjusting for both demographic variables and psychosocial working conditions, the adjusted OR of those working ≥60 hours was 3.25 (95% CI, 1.56–6.79). This study found that long working hours are significantly related to psychosocial stress responses among white-collar workers in one Korean company.