Background. The prevalence of and risk factors for abnormal anal cytology among men and women with human immunodeficiency virus (HIV) infection have not been extensively investigated. Methods. The Study to Understand the Natural History of HIV and AIDS in the Era of Effective Therapy (SUN study) is a prospective cohort study of HIV-infected patients in 4 US cities. Baseline questionnaires were administered and anal samples for cytology and human papillomavirus (HPV) detection and genotyping were collected. Results. Among 471 men and 150 women (median age, 41 years), 78% of participants were receiving combination antiretroviral therapy, 41% had a CD4+ cell count of ≥500 cells/μL, and 71% had an HIV RNA viral load of <400 copies/mL. The anal cytology results were as follows: 336 participants (54%) had negative results, 96 participants (15%) had atypical squamous cells, 149 participants (24%) had low-grade squamous intraepithelial lesions, and 40 participants (6%) had high-grade squamous intraepithelial lesions. In a multivariate analysis, abnormal anal cytology was associated with number of high-risk and low-risk HPV types (adjusted odds ratio [AOR] for both, 1.28; P < .001), nadir CD4 + cell count of <50 cells/μL (AOR, 2.38; P = .001), baseline CD4+ cell count of <500 cells/μL (AOR, 1.75; P = .004), and ever having receptive anal intercourse (AOR, 2.51; P < .001). Conclusion. HIV-infected persons with multiple anal HPV types or a nadir CD4+ cell count of <50 cells/μL have an increased risk for abnormal anal cytology. © 2010 by the Infectious Diseases Society of America. All rights reserved.