Abstract Background Few training opportunities are available for implementation practitioners; we designed the Practicing Knowledge Translation (PKT) to address this gap. The goal of PKT is to train practitioners to use evidence and apply implementation science in healthcare settings. The aim of this study was to describe PKT and evaluate participant use of implementation science theories, models, and frameworks (TMFs), knowledge, self-efficacy, and satisfaction and feedback on the course. Methods PKT was delivered to implementation practitioners between September 2015 and February 2016 through a 3-day workshop, 11 webinars. We assessed PKT using an uncontrolled before and after study design, using convergent parallel mixed methods. The primary outcome was use of TMFs in implementation projects. Secondary outcomes were knowledge and self-efficacy across six core competencies, factors related to each of the outcomes, and satisfaction with the course. Participants completed online surveys and semi-structured interviews at baseline, 3, 6, and 12 months. Results Participants (n = 15) reported an increase in their use of implementation TMFs (mean = 2.11; estimate = 2.11; standard error (SE) = 0.4; p = 0.03). There was a significant increase in participants’ knowledge of developing an evidence-informed, theory-driven program (ETP) (estimate = 4.10; SE = 0.37; p = 0.002); evidence implementation (estimate = 2.68; SE = 0.42; p