OBJECTIVES: Objectively measured sedentary behavior (SB) on weekdays and weekends has been mainly assessed in white-collar workers, while data in blue-collar workers are sparse. Therefore, this study presented the difference in accelerometer-measured SB levels between weekdays and weekends, stratified by white- and blue-collar occupations. METHODS: This study was a sub-analysis of accelerometer data from 73 workers (31 blue-collar and 42 white-collar) at a Japanese manufacturing plant. SB was defined as ≤1.5 metabolic equivalents estimated using an accelerometer, and compared between weekdays and weekends by using mixed models adjusted for confounders. The proportion of workers who sat for ≤8 h/day on weekdays and weekends were compared using McNemar's test. RESULTS: In white-collar workers, SB time on weekdays was significantly longer than that on weekends (598 vs 479 min/day, P < .001). In blue-collar workers, there was no significant difference in SB time between weekdays and weekends (462 vs 485 min/day, P = .43). The proportion of workers who achieved the recommended SB levels (≤8 h) was only 4.8% for white-collar workers on weekdays and 54.8% on weekends (P = .04), while that of blue-collar workers was 45.2% and 58.1% respectively (P > .99). CONCLUSIONS: White-collar workers were exposed to significantly longer SB time on weekdays than on weekends, which was not the case in blue-collar workers. It may be rather challenging for white-collar workers to limit their SB time to the level recommended by the latest guidelines for better health, especially on weekdays.