Abstract: Objective To understand the acceptability of female condom use and explore its influencing factors among HIV sero-discordant couples. Methods A prospective observational study was adopted to investigate the attitude, experience, and willingness to use female condom before and after 3 months among HIV sero-discordant couples in three cities of Shaoyang, Leshan, and Meishan through log records and questionnaire survey from May to December 2013. Results A total of 89 pairs of HIV sero-discordant couples were investigated. The mean age of HIV positive spouses and HIV negative spouses were (38.5±8.6) and (37.6±8.3) years, respectively. Of all the participants, 41.0% (73/178) had schooling level at junior middle school. The main job was the peasant (42.1%; 75/178) and the family income was between 2 000 and 2 999 Yuan (32.6%; 29/89). Among the couples, 66.3% (59/89) were husbands with HIV infection and 86.5% (77/89) were infected with HIV through heterosexual contact. Before enrollment of the study 44.9% (40/89) had sexual behaviors for 2-3 times a month, 86.5% (64/74) consistently used the condom in past 3 months before the study period. During the study period, 85.4% of them used 9 or more female condoms and 58.4% were willing to accept and continue to use the female condom after the study. Multivariate logistic regression showed that previous experience of compulsive sexual behavior (OR: 5.82), female spouse infected with HIV (OR: 3.79), applying lubricant in the penis (OR: 3.57), fully understanding the techniques after training (OR: 3.315), marriage period of 20 years or more (OR: 2.85),and experience of negative event when using the female condom for the first time (OR:7.14)were the independent positive factors influencing the willingness to use the female condom. Conclusion HIV sero-discordant couples have a certain degree of willingness to accept female condom. The female condom can be taken as an effective measure to prevent the spread of HIV among discordant couples.