Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca 2+ currents (I Ca,L ) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I Ca,L in human atrial myocytes and in HL-1 cells. Basal I Ca,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α 1C subunit of L-type Ca 2+ channel (LCC α 1C ) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y 416 ) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I Ca,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I Ca,L in HL-1 cells by ∼69% and ∼83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I Ca,L and down-regulated LCC α 1C subunit levels. The depression of I Ca,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I Ca,L amplitudes through impairment of channel function, down-regulation of LCC α 1C subunit levels, and the activation of c-Src kinases in atrial myocytes.