HIV pre-exposure prophylaxis (PrEP) was FDA approved in 2012, but uptake remains low. To characterize what would facilitate health care providers’ increased PrEP prescribing, we conducted a 10-city, online survey of 525 primary care providers (PCPs) and HIV providers (HIVPs) to assess awareness, knowledge, and experience with prescribing PrEP; and, comfort with and barriers to PrEP-related activities. Fewer PCPs than HIVPs had heard of PrEP (76% vs 98%), felt familiar with prescribing PrEP (28% vs. 76%), or had prescribed it (17% vs. 64%). PCPs were less comfortable than HIVPs with PrEP-related activities such as discussing sexual activities (75% vs. 94%), testing for acute HIV (83% vs. 98%), or delivering a new HIV diagnosis (80% vs. 95%). PCPs most frequently identified limited knowledge about PrEP and concerns about insurance coverage as prescribing barriers. PCPs and HIVPs differ in needs that will facilitate their PrEP prescribing. Efforts to increase PrEP uptake will require interventions to increase the knowledge, comfort, and skills of providers to prescribe PrEP.