The online BeUpstanding™ program is an eight-week workplace-delivered intervention for desk-based workers to raise awareness of the benefits of sitting less and moving more and build a supportive culture for change. A workplace representative (the "champion") delivers the program, which includes a workshop where teams collectively choose their sit less/move more strategies. A toolkit provides the champion with a step-by-step guide and associated resources to support program uptake, delivery, and evaluation. Here we report on the main findings from the Australian national implementation trial of BeUpstanding. Recruitment (12/06/2019 to 30/09/2021) was supported by five policy and practice partners, with desk-based work teams from across Australia targeted. Effectiveness was measured via a single arm, repeated-measures trial. Data were collected via online surveys, toolkit analytics, and telephone calls with champions. The RE-AIM framework guided evaluation, with adoption/reach (number and characteristics); effectiveness (primary: self-reported workplace sitting time); implementation (completion of core components; costs); and, maintenance intentions reported here. Linear mixed models, correcting for cluster, were used for effectiveness, with reach, adoption, implementation, and maintenance outcomes described. Of the 1640 website users who signed-up to BeUpstanding during the recruitment period, 233 were eligible, 198 (85%) provided preliminary consent, and 118 (50.6%) champions consented and started the trial, with 94% (n = 111 champions) completing. Trial participation was from across Australia and across industries, and reached 2,761 staff, with 2,248 participating in the staff survey(s): 65% female; 64% university educated; 17% from a non-English speaking background. The program effectively changed workplace sitting (-38.5 [95%CI -46.0 to -28.7] minutes/8-hour workday) and all outcomes targeted by BeUpstanding (behaviours and culture), with small-to-moderate statistically-significant effects observed. All participating teams (n = 94) completed at least 5/7 core steps; 72.4% completed all seven. Most champions spent $0 (72%) or >$0-$5 (10%) per team member; most (67/70 96%) intended to continue or repeat the program. BeUpstanding can be adopted and successfully implemented by a range of workplaces, reach a diversity of staff, and be effective at creating a supportive culture for teams of desk-based workers to sit less and move more. Learnings will inform optimisation of the program for longer-term sustainability. ACTRN12617000682347.