Background Training lay health workers is a critical strategy for HIV pre-exposure prophylaxis (PrEP) implementation, but little is known about effective approaches to conducting and evaluating such trainings. We evaluated the effect of the PrEP Diffusion Training, a one-day participatory training informed by the Diffusion of Innovation theory, on knowledge, PrEP stigma, implementation behavior, and diffusion of PrEP innovation among lay HIV workers in China. Methods Nine PrEP Diffusion Trainings were delivered to 220 lay HIV workers. We conducted multi-timepoint surveys to assess acceptability, appropriateness, and preliminary effects of the training on participants' knowledge, PrEP stigma, determinants of implementation behavior, and diffusion of PrEP information. We used an observation-based checklist to evaluate implementation delity. Results All trainings were delivered with high delity to the standardized manual, with little variability across sites. Acceptability and appropriateness of training were high. PrEP knowledge score signi cantly increased by 65% from pre-training to post-training, and remained high one month after the training. We observed a signi cant decrease in PrEP stigma and a positive in uence on determinants of implementation behaviors. All participants surveyed one-month after the training reported having shared PrEP information in their social networks, and 43% reported integrating PrEP education into their routine HIV prevention work on a regular basis. Conclusions The PrEP Diffusion Training is a practical and effective strategy to build lay HIV workers' capacity to participate in PrEP implementation. Longer-term follow up post training would be ideal to further assess actual PrEP uptake and sustained effects of intervention on PrEP implementation.