Persistent gastrointestinal inflammation, a hallmark of progressive HIV/SIV infection, causes disruption of the gastrointestinal epithelial barrier, microbial translocation, and generalized immune activation/inflammation driving AIDS progression. Apart from protein regulators, recent studies strongly suggest critical roles for microRNAs (miRNAs) in regulating and managing certain aspects of the inflammatory process. To examine their immunoregulatory role, we profiled miRNA expression in the colon from 12 chronic SIV-infected and 4 control macaques. After applying multiple comparisons correction, 10 (3 upregulated and 7 downregulated) miRNAs showed differential expression. Most notably, miR-34a showed significant upregulation in both epithelial and lamina propria leukocyte (LPL) compartments. Intense γH2A.X expression in colonic epithelium and LPLs confirmed the contribution of DNA damage response in driving miR-34a upregulation. SIRT1 mRNA and protein decreased significantly in both colonic epithelium and LPLs. Luciferase reporter assays validated rhesus macaque SIRT1 as a direct miR-34a target. Decreased SIRT1 expression was associated with constitutively enhanced expression of the transcriptionally active form of the p65 (acetylated on lysine 310) subunit of NF-κB exclusively in the LPL compartment. The intensity and number of acetylated p65(+) cells was markedly elevated in LPLs of chronically SIV-infected macaques compared with uninfected controls and localized to increased numbers of IgA(+) and IgG(+) plasma cells. These findings provide new insights into the potential role of the miR-34a-SIRT1-p65 axis in causing hyperactivation of the intestinal B cell system. Our results point to a possible mechanism where the normal immunosuppressive function of SIRT1 is inhibited by elevated miR-34a expression resulting in constitutive activation of acetylated p65 (lysine 310).