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INTRODUCTION: Thus far, the reasons for increasing HIV prevalence in northern and eastern Indian states are unknown. We investigated the role of male out-migration in the spread of human immunodeficiency virus (HIV) infection through a case-control study in rural India. METHODS: Currently married men and women were recruited from HIV testing and treatment centers across seven selected districts with high rates of male out-migration in eastern and northern India in 2010 using a case-control study design. Case subjects (men: 595, women: 609) were people who tested HIV seropositive and control subjects (men: 611, women: 600) were those tested HIV seronegative. For each gender, we obtained adjusted odds ratios (AORs) and population attributable risks (PARs) for migration, and behavioral factors. RESULTS: For men, the prevalence of HIV was significantly higher among those with a migration history (AOR, 4.4); for women, the prevalence of HIV was higher among those with migrant husbands (AOR, 2·3). For both genders, the returned male migration (men: AOR, 3·7; women: AOR, 28) was significantly associated with higher prevalence of HIV infection. The PAR associated with male migration was higher for men (54.5%-68.6%) than for women (32·7%-56·9%) across the study areas. DISCUSSION: Male out-migration is the most important risk factor influencing the spread of HIV infection in rural areas with high out-migration rates, thereby emphasizing the need for interventions, particularly, for returned migrants and spouses of those migrants.