Background: The Web-based, evidence-informed BeUpstanding Champion Toolkit was developed to provide employers (via a “train-the-champion approach”) with resources and support to help in reducing prolonged sitting in their own desk-based workplace. As part of a five-phase research-to-dissemination process, this study reports on the evaluation of the beta (test) version of this toolkit (Phase 2). Objective: The objective of our study was to evaluate (1) the implementation of the toolkit by workplace champions and (2) the impact of the toolkit on sitting (primary outcome), standing, and moving; use of activity-promoting strategies; knowledge and attitudes; and indicators of health and work performance. Methods: An implementation study using a pre-post design was conducted in 7 desk-based workplaces in Australia (September 2015 to May 2016), with work teams (one per workplace) purposively recruited to ensure representation across a range of sectors (white- or blue-collar), organizational sizes (small or medium or large), and locations (metropolitan or regional). All staff within participating teams were invited to participate in the relevant toolkit activities. Implementation outcomes (time commitment required by champions and toolkit activities completed) were collected from each champion via telephone interviews. Changes in impact outcomes, measured via a Web-based questionnaire completed by employees at baseline and 3 months postimplementation, were assessed using mixed models, correcting for clustering. Results: Champions reported a 30-60 minutes per week time commitment to the toolkit activities. All teams formed a wellbeing committee and sent the staff surveys at both time points; most champions held a staff consultation workshop (6/7), identified team-level strategies within that workshop (5/7), used the communication resources provided within the toolkit (emails, posters; 6/7), and completed the action plan (5/7). In total, 52% (315 of ≈600) employees participated in at least one survey and 97 (16%) participated in both. At follow-up, there was a significant (P<.05) reduction in self-reported workplace sitting time compared to baseline (−6.3%, 95% CI −10.1 to −2.5; n=85) equating to ≈30 minutes per workday. Significant benefits were also observed for the use of activity-promoting strategies, with small, nonsignificant changes observed for knowledge and attitudes and indicators of health and work performance. Conclusions: The beta version of the BeUpstanding Champion Toolkit was feasible to implement and effective in reducing self-reported workplace sitting across a broad range of desk-based workplaces. The next phase (Phase 3) will build on these findings to optimize the toolkit for wider-scale implementation and longer term evaluation.