Transgender women (TW) and men who have sex with men (MSM) in Kenya are disproportionately affected by human immunodeficiency virus (HIV) and would benefit substantially from pre-exposure prophylaxis (PrEP). We conducted focus group discussions (FGDs) with healthcare providers (HCPs) and TW/MSM leadership and in-depth interviews (IDIs) with PrEP-experienced MSM and TW to learn about perceived and actual barriers to PrEP programming. Eleven HCP and 10 TW/MSM leaders participated in FGDs before PrEP roll-out (January 2018) and 12 months later. Nineteen PrEP end-users (11 MSM and 8 TW) participated in IDIs. Topic guides explored PrEP knowledge, HIV acquisition risk, gender identity, motivation for PrEP uptake and adherence and PrEP-dispensing venue preferences. Braun and Clarke thematic analysis was applied. Four themes emerged: limited preparedness of HCPs to provide PrEP to TW and MSM, varied motivation for PrEP uptake and persistence among end users, lack of recognition of TW by HCPs and suggestions for PrEP programming improvement from all stakeholders. Providers’ reluctance to prescribe PrEP to TW and distrust of TW towards providers calls for interventions to improve the capacity of service environments and staff HIV preventive care. Alternative locations for PrEP provision, including community-based sites, may be developed with TW/MSM leaders.