MicroRNAs (miRNAs) act as important post-transcriptional regulators of gene expression by targeting the 3'-untranslated region of their target genes. Altered expression of miR-16 is reported in human ulcerative colitis (UC), but its role in the development of the disease remains unclear. Adenosine through adenosine A2a receptor (A2aAR) could inhibit nuclear factor-kappaB (NF-κB) signaling pathway in inflammation. Here we identified overexpression of miR-16 and down-regulation of A2aAR in the colonic mucosa of active UC patients. We demonstrated that miR-16 negatively regulated the expression of the A2aAR at the post-transcriptional level. Furthermore, transfection of miR-16 mimics promoted nuclear translocation of NF-κB p65 protein and expression of pro-inflammatory cytokines, IFN-γ and IL-8 in colonic epithelial cells. Treatment with miR-16 inhibitor could reverse these effects in cells. The A2aAR-mediated effects of miR-16 on the activation of the NF-κB signaling pathway were confirmed by the A2aAR knockdown assay. Our results suggest that miR-16 regulated the immune and inflammatory responses, at least in part, by suppressing the expression of the A2aAR to control the activation of the NF-κB signaling pathway.