This study aims to clarify the relationships between length of overtime work and various stress responses using large-scale cross-sectional data of Japanese workers. This study's participants are 59,021 Japanese workers in 117 companies. Data was collected by self-reporting questionnaire. The Brief Job Stress Questionnaire was used to measure stress responses on six scales (i.e. lack of vigor, irritability, fatigue, anxiety, depression, and somatic responses). Length of overtime work hours were classified as 0-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and >80 hours/month. Multiple linear regression analyses were used to examine the association of stress responses with overtime while adjusting all possible confounders. In result, workers with longer overtime showed significantly higher irritability, fatigue, anxiety, depression, and somatic responses for both genders (p-for-trend <0.001), however, length of overtime was negatively associated with lack of vigor among men (p-for-trend <0.001). Men with 61-80 hours of overtime showed high fatigue with high vigor at the same time. Length of overtime was linearly associated with various stress responses, except for lack of vigor. Length of overtime shows linear associations with various psychosomatic stress responses. However, lack of vigor was not consistently associated with overtime. Male workers with 61-80 hours of monthly overtime were more likely to feel vigorous than workers with shorter overtime. However, potential longterm effects of such extreme overtime should not be underestimated and must be paid attention to.