T cells that express the γδ T-cell receptor, which recognize microbial or stress-induced antigens, represent a minority of blood T cells but constitute a major proportion of intraepithelial lymphocytes in the gastrointestinal mucosa. As microbial products have been shown to translocate from the gastrointestinal tract into circulation in chronically HIV/Simian immunodeficiency virus (SIV)-infected individuals, we conducted a study of Vδ1 and Vδ2 T-cell frequency, phenotype, and function in blood, spleen, lymph nodes, gastrointestinal mucosa, and bronchoalveolar lavage of uninfected and chronically SIVsmE543-infected rhesus macaques (RMs). We found: (1) SIV-associated inversion of Vδ1/Vδ2 T cells occurs in blood and in several tissues; (2) γδ T cells are not infected by SIV in vivo; (3) the Vδ1/Vδ2 inversion involves expansion of Vδ1 T cells; (4) expanded Vδ1 T cells are phenotypically and functionally different from Vδ1 T cells from uninfected RMs; and (5) the stimulus underlying expansion of Vδ1 T cells appears to be microbial translocation. These data highlight the importance of microbial translocation-induced immune activation in chronically infected individuals and provide new insights into an immune dysregulation phenomenon that is a hallmark of HIV/SIV infection. These findings may lead to novel therapeutic interventions that improve the immune responses against microbial antigens, and thus, decrease microbial translocation-induced immune activation.