Health care workers (HCWs) in sub-Saharan Africa are at a high risk of HIV infection from both sexual and occupational exposures. However, many do not seek HIV testing. This paper examines the acceptability of an unsupervised facility-based HIV self-testing (HIVST) intervention among HCWs and their partners and factors associated with uptake of HIVST among HCWs. HCWs in seven large Kenyan hospitals were invited to participate in pre-HIVST information sessions during which they were offered HIVST kits to take home for self-testing. A post-intervention survey was conducted among 765 HCWs. Forty-one percent attended the information session; of those, 89% took the HIVST kits and of those, 85% self-tested. Thirty-four percent of surveyed HCWs used the HIVST to test themselves. Of those who took the HIVST kit and had partners, 73% gave the kit to their partner and 86% of them indicated their partner self-tested. Factors positively associated with use of the HIVST on self were being female, being single, and being a HCW from Homa Bay Hospital (located in a high HIV prevalence area). HIVST is acceptable to HCWs and their partners. However, strategies are needed to increase HCWs attendance at pre-implementation information sessions.