A pharmacokinetic study evaluated the effect of masculinizing hormone therapy (MHT; i.e., testosterone enanthate 200 mg IM) on oral daily PrEP among 20 trans men in Thailand. Testosterone enanthane was administered at baseline and every 2 weeks until week 12 while oral daily emtricitabine/tenofovir alafenamide -based PrEP (200/25 mg) was prescribed from week 6 until the end of the study at week 16. Pharmacokinetic sampling was performed at week 12 (PrEP with MHT) and week 16 (PrEP without MHT) to assess plasma concentrations of emtricitabine (FTC), tenofovir alafenamide (TAF) and tenofovir (TFV). In addition, intracellular TFV-diphosphate (TFV-DP) and FTC triphosphate (FTC-TP) concentrations were assessed in peripheral blood mononuclear cells, rectal and cervical tissues. Geometric mean ratios (GMRs) (90% CI) of AUC0-24 and Cmax at week 12 and 16 (reference) were: TAF, 0.99 (0.80-1.21, p=0.93) and 1.00 (0.69-1.44, p=0.99); TFV, 1.03 (0.94-1.14, p=0.63) and 0.97 (0.84-1.43, p=0.64); FTC, 1.02 (0.99-1.05, p=0.48) and 0.97 (0.85-1.12, p=0.80). There were no statistically significant differences in median FTV-DP and FTC-TP concentrations in PBMCs (C24 TFV-DP, 564.2 vs 631.9 fmol/10^6 cells, p=0.77; C24 FTC-TP 3341.2 vs 3888.3 fmol/10^6 cells, p=0.60) and rectal tissue (FTV-DP, 53.4 vs 65.3 fmol/mg, p=0.51; FTC-TP, 7.6 vs 7.7 fmol/mg, p=0.51) between the two visits. However, both TFV-DP and FTC-TP concentrations in cervical tissue were significantly lower at week 12 and 16 (TFV-DP, 12.9 vs 20.6 fmol/mg, p=0.04; FTC-TP, 67.1 vs 120.4 fmol/mg, p=0.02). In conclusion, plasma TAF, TFV and FTC and intracellular TFV-DP and FTC-TP levels in PBMCs and rectal tissue were comparable when FTC/TAF-based PrEP was co-administered with MHT. However, TFV-DP and FTC-TP concentrations tended to be lower in cervical tissue. The clinical relevance is unknown.