Abstract: Objective To explore the factors influencing the loss to follow-up over the first five years among seronegative spouses of HIV-infected individuals in Dehong Prefecture, Yunnan Province. Methods A total of 1 110 HIV seronegative spouses were recruited in 2009, social-demographic characteristics and HIV/AIDS related information were collected, follow-up survey was conducted every year till the end of 2014.COX proportional hazards regression model was used to analyze risk factors of loss to follow-up. Results Among total 1 110 seronegative spouses recruited, HIV seroconvertion was developed in 37 subjects with a total incidence rate of 0.92 per 100 person years. 1 015(91.4%) had at least one follow-up visit, however, 227 lost to follow-up at the end of 2014, contributing to a rate of 5.66 per 100 person years. four subjects and 129 seropositive subjects died, 63 divorced. The retention rate was 58.7%(652/1 110) at 5 years. Of all sero-negative spouses who needed follow-up, 74.2% (652/879) performed follow-up visit. COX proportional hazards regression model showed that the risk factors of loss to follow-up were living in Ruili (HR=1.83,95% CI:1.16-2.87,P=0.009) or Mangshi (HR=1.90,95% CI:1.23-2.93,P=0.004), male (HR=1.74,95% CI:1.27-2.38,P=0.001) and having sero-positive spouses not under anti-retroviral therapy (ART) (HR=4.39,95% CI:3.21-6.02,P<0.001). Conclusions Living in Ruili or Mangshi, male and having seropositive spouses not under ART are risk factors of loss to follow-up. To maintain a high retention rate, the institute of HIV/AIDS prevention and control in Dehong prefecture should pay more attention to these influencing factors in the future work.