Background Emerging evidence suggests that sedentary behaviour may be adversely associated with physical health, but few studies have examined the association with mental well-being. Purpose This study examined the association of four non-occupational sedentary behaviours, individually and in total, with mental well-being in employed adults. Methods Baseline data from the evaluation of Well@Work, a national workplace health promotion project conducted in the UK, were used. Participants self-reported sitting time whilst watching television, using a computer, socialising and travelling by motorised transport. Mental well-being was assessed by the 12-item version of the general health questionnaire. Analyses were conducted using multiple linear regression. Results In models adjusted for multiple confounders, TV viewing, computer use and total non-occupational sitting time were adversely associated with general health questionnaire-12 assessed mental well-being in women. Computer use only was found to be adversely associated with mental well-being in men. Conclusion Sedentary behaviour may be adversely associated with mental well-being in employed adults. The association may be moderated by gender.