Background: Sitting, particularly in prolonged, unbroken bouts, is widespread within the office workplace, yet few interventions have addressed this newly-identified health risk behaviour. This paper describes the iterative development process and resulting intervention procedures for the Stand Up Australia research program focusing on a multi-component workplace intervention to reduce sitting time. Methods: The development of Stand Up Australia followed three phases. 1) Conceptualisation: Stand Up Australia was based on social cognitive theory and social ecological model components. These were operationalised via a taxonomy of intervention strategies and designed to target multiple levels of influence including: organisational structures (e.g. via management consultation), the physical work environment (via provision of height-adjustable workstations), and individual employees (e.g. via face-to-face coaching). 2) Formative research: Intervention components were separately tested for their feasibility and acceptability. 3) Pilot studies: Stand Up Comcare tested the integrated intervention elements in a controlled pilot study examining efficacy, feasibility and acceptability. Stand Up UQ examined the additional value of the organisational- and individual-level components over height-adjustable workstations only in a three-arm controlled trial. In both pilot studies, office workers' sitting time was measured objectively using activPAL3 devices and the intervention was refined based on qualitative feedback from managers and employees. Results: Results and feedback from participants and managers involved in the intervention development phases suggest high efficacy, acceptance, and feasibility of all intervention components. The final version of the Stand Up Australia intervention includes strategies at the organisational (senior management consultation, representatives consultation workshop, team champions, staff information and brainstorming session with information booklet, and supportive emails from managers to staff), environmental (height-adjustable workstations), and individual level (face-to-face coaching session and telephone support). Stand Up Australia is currently being evaluated in the context of a cluster-randomised controlled trial at the Department of Human Services (DHS) in Melbourne, Australia. Conclusions: Stand Up Australia is an evidence-guided and systematically developed workplace intervention targeting reductions in office workers' sitting time. © 2014 Neuhaus et al.; licensee BioMed Central Ltd.