Purpose: The aim of this study was to clarify the influence of shift work on serum total cholesterol (T-Cho) levels according to body mass index (BMI) at entry in Japanese male workers.Methods: A 14-year prospective cohort study was conducted in day workers (n = 4079) and alternating shift workers (n = 2807) who received annual health check-ups between 1991 and 2005 in a Japanese steel company. The association between job schedule and increases in T-Cho was investigated using multivariate pooled logistic regression analyses with age, BMI, lifestyle (smoking habit, drinking habit, habitual exercise), and the results of blood chemistries (creatinine, glycosylated hemoglobin A1c, aspartic aminotransferase, γ-glutamyl transpeptidase, uric acid) serving as covariates in those who were not overweight (BMI < 25 kg/m2, n = 5082) and overweight (BMI ≥ 25 kg/m2, n = 1804) at entry, respectively. The endpoints in the study were either a 20%, 25%, 30%, 35%, 40%, or 45% increase in T-Cho during the period of observation compared to T-Cho at entry.Results: In subjects who were not overweight at entry, alternating shift work was associated significantly with five serum T-Cho endpoints (≥20%: odds ratio [OR] = 1.15, 95% confidence interval [CI)] = 1.05, 1.26; ≥25%: OR = 1.17, 95% CI = 1.05, 1.31; ≥35%: OR = 1.24, 95% CI = 1.05, 1.46; ≥40%: OR = 1.30, 95% CI = 1.06, 1.61; ≥45%: OR = 1.31, 95% CI = 1.01, 1.71). However, alternating shift work was not associated with any of the six T-Cho endpoints in subjects who were overweight at entry.Conclusions: In the present study, shift work was shown to be a potential risk factor for increased T-Cho in non-overweight Japanese male workers. However, we did not obtain a consistent association between shift work and an increase in T-Cho levels in overweight subjects. The results suggest that the effect of shift work on lipid regulation may be influenced by BMI.