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Antiretroviral therapy (ART) and condom use have been proven to reduce the risk of sexual transmission of human immunodeficiency virus (HIV) among HIV sero-different couples, but its full implementation remains a challenge. This study aims to assess HIV seroconversion rate of HIV-negative spouse and its associated risk factors among HIV sero-different couples in rural China.An open cohort of HIV sero-different couples enrolled in 30 counties in China between October 1, 2010, and September 30, 2012, and followed-up to December 31, 2012, was constructed retrospectively. A nested case-control study of risk factors of HIV seroconversion among sero-different couples was conducted in April and May of 2013, based on the open cohort. Sero-different couples with the HIV-negative spouse seroconverting at least 3 months after the previous negative diagnosis during cohort observation period were labeled as "case couples". The "control couples" were selected randomly from the same cohort that did not have the HIV-negative spouse seroconversion during the same period. The "case couples" and "control couples" were matched on gender, age, and region of residence. Sexual behaviors among HIV sero-different couples before and after the index spouses notifying their HIV infection status to their HIV-negative spouses were collected via face-to-face interview. Univariate and multivariate logistic regression models were used to assess factors associated with HIV seroconversion among HIV sero-different couples.Of 4481 HIV sero-different couples, a total of 53 seroconversions were observed within 5218 person-years of follow-up. The incidence rate was 1.02 (95%CI: 0.76-1.33) per 100 person-years. Forty "case couples" confirmed HIV-negative spouse seroconversions infected via marital sexual transmission, were matched to 80 "control couples". Of the 120 couples, 81(67.5%) were receiving ART, and 70 (58.3%) reported consistently used condoms during intercourse after the index spouse was diagnosed HIV infection. Multivariate conditional logistic regression analysis showed that the desire to conceive a child (OR = 5.18, 95% CI: 1.19-22.58) significantly increased the odds of HIV seroconversion. Protective factors of spousal HIV seroconversion were currently receiving ART (OR = 0.09, 95% CI: 0.01-0.67) and consistent condom use (OR = 0.05, 95% CI: 0.01-0.28).Intention to conceive a child is the most important risk factor for HIV seroconversion among sero-different couples. Specific efforts on scientific use of ART to assist sero-different couples to achieve their wish to conceive a healthy child are needed to minimize the risk of HIV transmission.