Objectives: To examine (1) the inter-relationships between socio-economic status (SES), physical activity, three different domains of sitting time (weekday, weekend day and leisure-time sitting), and being overweight or obese (body mass index≥25 kg/m2); and (2) the potential mediation effects of sitting time in the relationship between socio-economic factors and being overweight or obese in working Australian adults. Design: Observational epidemiological study. Subjects: One thousand forty eight working adults. Using a multistage sampling design on neighbourhood SES, participants were from high and low SES neighbourhoods of an Australian capital city. Measurements: Neighbourhood SES was assessed using census data; individual SES was based on self-reported educational attainment and household income. There were three sitting time variables: sitting time on weekdays, weekend days and in leisure time. Overweight and obesity were determined using self-reported body weight and height. Results: Gender, age, neighbourhood SES, education, working hours and physical activity were independently associated with weekday, weekend day and leisure-related sitting time. With the exception of education and working hours, these variables were also independently associated with being overweight or obese. Leisure-time sitting was found to be a mediator in the relationships between gender, education and being overweight or obese. Conclusion: Strategies to promote less sitting in leisure time are required to combat overweight and obesity in Australian adults, especially among those from low SES neighbourhoods, and among those with high levels of education and income who work long hours.