Qualitative studies identified barriers and facilitators associated with work-related sedentary behaviour. The objective of this study was to determine common perceived barriers and facilitators among office workers, assess subgroup differences, and describe sedentary behaviour. From two Swedish companies, 547 office workers (41 years (IQR = 35–48), 65% women, 66% highly educated) completed questionnaires on perceived barriers and facilitators, for which subgroup differences in age, gender, education, and workplace sedentary behaviour were assessed. Sedentary behaviour was measured using inclinometers (n = 311). The most frequently reported barrier was sitting is a habit (67%), which was reported more among women than men (X2 = 5.14, p = 0.03) and more among highly sedentary office workers (X2 = 9.26, p < 0.01). The two other most reported barriers were that standing is uncomfortable (29%) and standing is tiring (24%). Facilitators with the most support were the introduction of either standing- or walking-meetings (respectively 33% and 29%) and more possibilities or reminders for breaks (31%). The proportion spent sedentary was 64% at the workplace, 61% on working days, and 57% on non-working days. This study provides a detailed understanding of office workers’ ideas about sitting and means to reduce sitting. We advise to include the supported facilitators and individualized support in interventions to work towards more effective strategies to reduce sedentary behaviour.