This study examined whether short sleep duration, physical activity and time spent sitting each day mediated the association between long work hours and body mass index (BMI). Participants included 16,951 middle aged Australian adults who were employed in full time work (i.e. ≥35 h a week). Data on BMI, sleep duration, work hours and other health and demographic variables were obtained through a self-report questionnaire. A multiple mediation model was tested whereby sleep duration, physical activity and amount of time spent sitting were entered as potential mediators between work hours and BMI. The results demonstrated that short sleep partially mediated the association between long work hours and increased BMI in males. In females, long work hours were indirectly related to higher BMI through short sleep. The results provide some support for the hypothesis that long work hours could contribute to obesity via a reduction in sleep duration; this warrants further investigation in prospective studies.