Background: Prolonged sitting is prevalent in the workplace and is associated with adverse health markers. Purpose: Investigate the effects of point-of-choice (PoC) prompting software, on the computer used at work (PC), to reduce long uninterrupted sedentary periods and total sedentary time at work. Design: Assessor-blinded, parallel group, active-controlled randomized trial. Setting/participants: A convenience sample of office workers from Glasgow, United Kingdom. Data were collected April to June 2010, and analyzed October 2010 to June 2011. Intervention: The education group (n=14) received a brief education session on the importance of reducing long sitting periods at work. The PoC group (n=14) received the same education along with prompting software on their PC for 5 workdays, which reminded them to stand up every 30 minutes. Main outcome measures: Sitting time was measured objectively using the activPAL™ activity monitor for 5 workdays at baseline and 5 workdays during the intervention. The number and time spent sitting in events >30 minutes' duration were the main outcome measures. Results: At baseline, participants spent 5.7±1.0 hours/day (76%±9%) of their time at work sitting. Of that time, 3.3±1.3 hours/day was spent sitting in 3.7±1.4 events >30 minutes. There was a significant difference between the groups in the change (intervention to baseline) of both the number (ANCOVA; -6.8%, p=0.014) and duration (-15.5%, p=0.007) of sitting events >30 minutes. During the intervention, compared with baseline, the PoC group reduced the number (paired t-test; -0.11 events/hour, p=0.045) and duration (-12.2%, p=0.035) of sitting events >30 minutes. However, there was no significant difference in total sitting time between groups (-4.4%, p=0.084). Conclusions: Point-of-choice prompting software on work computers recommending taking a break from sitting plus education is superior to education alone in reducing long uninterrupted sedentary periods at work. Trial registration: This trial was registered at ClinicalTrials.gov NCT01628861.