Increasing evidence suggests that miRNAs are widely dysregulated in ulcerative colitis (UC), potentially affecting UC pathogenesis, diagnosis, and therapy. microRNA (miR) -206 has been reported to be upregulated in UC; however, its function and role in UC remain unknown. Here, we elucidate the function of miR-206 in the pathogenesis of UC. In patients with active-UC, miR-206 and adenosine A3 receptor (A3AR) levels were significantly upregulated and downregulated, respectively, and were inversely correlated. A3AR was expressed in the colon mucosa (particularly in colon epithelial-cell membranes). In HT-29 cells, miR-206 downregulated A3AR mRNA/protein expression by directly targeting the A3AR 3'-UTR; miR-206 overexpression and knockdown respectively increased and decreased TNF-α-induced nuclear NF-κB/p65, p-IκB-α, IKKα, p-IKKα and IL-8/IL-1β secretion. However, A3AR-siRNA reversed the miR-206 inhibitory effect. Furthermore, miR-206 increased dextran sodium sulphate-induced colitis severity (i.e., increased bodyweight loss, DAI score, colon shrinkage, and MPO activity), which was partially ameliorated by miR-206-antagomir treatment. miR-206-agomir treatment potently suppressed A3AR expression and increased NF-κB signalling and downstream cytokine (TNF-α/IL-8/IL-1β) expression in the mouse colon, in contrast to miR-206-antagomir administration. Taken together, our results demonstrated that miR-206 has a proinflammatory role in UC by downregulating A3AR expression and activating NF-κB signalling.