Intimate partners of people living with HIV are at risk of HIV infection. We assessed the acceptability of female condom use among 89 married, heterosexual, HIV-serodiscordant couples from Sichuan and Hunan provinces in China for this prospective observational cohort study. Participants used female condoms for 3 months, reporting use and attitudes in written logs and questionnaires. At the end of the study, 58.4% of couples expressed willingness to continue using female condoms. Factors associated with willingness to use female condoms were (a) the female partner reporting having experienced forced sex by the male partner, (b) applying a lubricant to the penis, (c) understanding the correct application method, (d) being married more than 20 years, and (e) experiencing no difficulty during the first use. Most HIV-serodiscordant couples found female condoms to be acceptable. Increasing access to female condoms could be an acceptable alternative barrier method to male condoms for preventing HIV transmission.