Connexin 43 (Cx43) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the effect of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, on Cx43 expression and activity and determine the intracellular signaling pathways. Cx43 protein and mRNA levels were assayed using immunofluorescence, real‐time PCR, and western blot. We found that increased MIF and extracellular regulated protein kinases (ERK) expression was accompanied by a significant reduction in Cx43 protein expression in atrial tissues from patients with AF compared with those with sinus rhythm. In cultured atrium‐derived myocytes (HL‐1 cells), mouse recombinant‐MIF (rMIF, 20 or 40 nM, 24 h) down‐regulated gene and protein expression of Cx43 in a concentration‐dependent manner. U0126, a specific inhibitor of mitogen‐activated protein kinase kinase (MAPKK) could reverse the decrease in expression of Cx43 protein induced by rMIF. Further studies revealed that rMIF (40 nM, 15, 30, and 45 min) was able to stimulate phospho‐Erk1/2 (Thr202/Tyr204) production in a time‐dependent manner. These results suggest that MIF is involved in the pathogenesis of AF, probably by down‐regulating the protein and gene expression of Cx43 via ERK1/2 kinase activation. Our findings represent a potential pathogenic mechanism in AF. This article is protected by copyright. All rights reserved.