MicroRNA (miR)-142 is up-regulated in the brain in HIV and SIV encephalitis (SIVE). We identified the cell types where miR-142 is up-regulated and its relevant downstream target. Fluorescent in situ hybridization combined with immunofluorescent labeling revealed that miR-142-3p and -5p are expressed within hippocampal neurons and myeloid cells in SIVE. Sirtuin1 (SIRT1) was predicted as a potential miR-142 target by analysis of its 3'-UTR and bioinformatic analysis of factors linked to altered hippocampal gene expression profile in SIVE. Overexpression of pre-miR-142 in HEK293T cells led to a 3.7-fold decrease in SIRT1 protein level. Examination of the individual effects of miR-142-5p and miR-142-3p through overexpression and inhibition studies revealed that significant effects on SIRT1 occurred only with miR-142-5p. Luciferase reporter assays revealed a 2.3-fold inhibition of expression due to interaction of miR-142 with the SIRT1 3'-UTR, mutation analysis revealed that only the miR-142-5p target site was active. MiR-142 expression in primary human neurons led to a small (1.3-fold) but significant decrease in SIRT1 protein level. Furthermore, qRT-PCR revealed up-regulation of miR-142-3p (6.4-fold) and -5p (3.9-fold) and down-regulation of SIRT1 (33-fold) in macrophages/microglia from animals with SIVE. We have therefore elucidated a miR-mediated mechanism of regulation of SIRT1 expression in SIVE.