Rationale: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca ²⁺ regulates gene expression, but the nature and significance of nuclear Ca ²⁺ -changes in AF are largely unknown. Objective: To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca ²⁺ ([Ca ²⁺ ] _Nuc ) and CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase-II)-related signaling. Methods and Results: Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca ²⁺ ] _Nuc and cytosolic [Ca ²⁺ ] ([Ca ²⁺ ] _Cyto ) were recorded via confocal microscopy. Diastolic [Ca ²⁺ ] _Nuc was greater than [Ca ²⁺ ] _Cyto under control conditions, while resting [Ca ²⁺ ] _Nuc was similar to [Ca ²⁺ ] _Cyto ; both diastolic and resting [Ca ²⁺ ] _Nuc increased with AF. IP ₃ R (Inositol-trisphosphate receptor) stimulation produced larger [Ca ²⁺ ] _Nuc increases in AF versus control cardiomyocytes, and IP ₃ R-blockade suppressed the AF-related [Ca ²⁺ ] _Nuc differences. AF upregulated nuclear protein expression of IP ₃ R1 (IP ₃ R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca ²⁺ ] _Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP ₃ R knockdown with short-interfering RNA directed against IP ₃ R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP ₃ R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP ₃ R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP ₃ R1 upregulation and nuclear diastolic Ca ²⁺ -loading. Conclusions: AF increases atrial-cardiomyocyte nucleoplasmic [Ca ²⁺ ] by IP ₃ R1-upregulation involving miR-26a, leading to enhanced IP ₃ R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.