Background— Enhanced sarcoplasmic reticulum Ca ²⁺ -leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3′-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca ²⁺ -leak. Methods and Results— Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3′-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25 ^−/− mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca ²⁺ -spark frequency and total sarcoplasmic reticulum Ca ²⁺ -leak were increased in atrial myocytes of miR-106b-25 ^−/− mice. Telemetry ECG recordings revealed that miR-106b-25 ^−/− mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca ²⁺ -release and AF susceptibility in miR-106b-25 ^−/− mice were abolished by the RyR2 blocker K201. Conclusions— These results suggest that miR-106b-25 cluster–mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.