The gastrointestinal tract (GIT) is a major target of infection with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Chronic GIT disease and inflammation are common sequelae to HIV/SIV infection. Nonetheless, the molecular mechanisms that cause and maintain GIT dysfunction remain unclear. We investigated the contribution of CCAAT/enhancer-binding protein beta (C/EBPbeta) to GIT disease and viral replication in jejunum and colon collected at necropsy from 12 SIV-infected (group 1), or 10 uninfected macaques with chronic diarrhea (group 2), and 9 uninfected control macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more severe lesions in the jejunum. C/EBPbeta gene expression increased significantly in colon of groups 1 and 2 and in jejunum of only group 1 macaques compared with controls. In group 1 animals, CEBPbeta expression was localized predominantly to macrophages and occasionally lymphocytes. Chromatin immunoprecipitation assays confirmed the binding of C/EBPbeta and p65 to the SIV long terminal repeat region in colonic lamina propria cells, suggesting a mechanistic link between inflammation and activation of viral replication in vivo. This is the first in vivo study describing the transcriptional changes and immunophenotypic localization of C/EBPbeta in the GIT of SIV-infected macaques. More importantly, these data provide a molecular mechanism for persistent inflammation and immune activation leading to increased SIV burden and GIT pathology in SIV-infected macaques and perhaps HIV-infected individuals.