Objective To investigate the acceptability and influencing factors of pre-exposure prophylaxis (Pr-EP) for human immunodeficiency virus (HIV) among female sex workers (FSWs) in Sichuan, Xinjiang and Guangxi of China. Methods FSWs selected from Sichuan, Xinjiang and Guangxi were surveyed with questionnaires. A total of 1 478 questionnaires were completed, among which 1 469 (99.39%) were effective. The survey mainly concerned with the awareness and acceptability of FSWs on Pr-EP. Results After introduction to Pr-EP by the investigators, under the precondition that Pr-EP was effective and safe, 997(67.9%) FSWs accepted Pr-EP, and there was no significant difference in the acceptability among FSWs in three regions (P>0.05). Univariate χ² analysis and Logistic regression analysis revealed that self-evaluation of risks (OR=1.776, P=0.005), regular condom use in sex service (OR=1.346, P=0.000), frequency of condom use during the latest three times of sex service (OR=1.519, P=0.003), drug use to prevent sexually-transmitted disease (OR=1.185, P=0.025), refusal of having sex with those without condom use (OR=1.479, P=0.009) and advising peers to use Pr-EP (OR=5.618, P=0.000) were influencing factors of acceptability of Pr-EP. FSWs mainly concerned of the security, effectiveness and cost of Pr-EP. Conclusion FSWs with regular condom use are more likely to accept Pr-EP. Pr-EP related knowledge should be well exposed among FSWs to increase the awareness and acceptability. Peer education may be a better way to improve the acceptability of Pr-EP.