Human papillomavirus (HPV) is the main etiologic agent of lower genital tract cancers. The natural history of HPV infection and the immune response to HIV/HPV co-infection, particularly in the anal mucosa, is poorly understood. The aim was evaluate the in situ immune response in anorectal biopsies from HIV-infected patients. A total of 114 biopsies were analyzed by Tissue Micro-Array: 15 were from HIV-negative individuals with normal squamous epithelium and 99 from HIV-positive individuals (21 normal squamous epithelium, 39 with anal intra-epithelial lesion grade 1 and 39 with anal intra-epithelial lesion grade 2/3). PCR and sequencing were used to identify HPV DNA. Staining for CD4, CD8, Foxp3+, T-bet and IL-10 were analyzed via immunohistochemistry. HIV-positive patients with AIN 2/3 showed a lower number of CD4+ cells (< 50 cells/mm3) compared to HIV negative subjects (P = 0.01). HIV infected individuals showed a higher expression of FoxP3+ and IL-10 that correlated with the severity of the lesion (P = 0.002). A positive coefficient correlation was found between FoxP3+ and IL-10 (r = 0.34; P = 0.027). HPV DNA was detected in 93.4% (101/107) of the samples and the most common types were HPV 16 (26.9%), followed by HPV 6 (15.7%), HPV 59 (13%) and HPV 18 (10.2%). Our results showed a strong association between the increased T-reg cells and IL-10 expression in HIV-positive patients with AIN 2/3. HPV 16 was the most prevalent type. Our study suggests that the immune regulatory in situ profile may favor HPV persistence in HIV-positive individuals. Further in situ studies should be done in order to elucidate the development of anal cancer in HPV/HIV co-infected individuals.