Background Atrial fibrillation (AF) is the most common sustained arrhythmia, and pulmonary veins (PVs) play a critical role in triggering AF. Angiotensin (Ang)-(1-7) regulates calcium (Ca2+) homeostasis and also plays a critical role in cardiovascular pathophysiology. However, the role of Ang-(1-7) in PV arrhythmogenesis remains unclear. Materials and methods Conventional microelectrodes, whole-cell patch-clamp, and the Fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca2+ in isolated rabbit PV preparations, and in single isolated PV cardiomyocytes, before and after administration of Ang-(1-7). Results Ang (1-7) concentration-dependently (0.1, 1, 10, and 100 nM) decreased PV spontaneous electrical activity. Ang-(1-7) (100 nM) decreased the late-sodium (Na+), L-type Ca2+, and Na+-Ca2+ exchanger currents, but did not affect the voltage-dependent Na+ current in PV cardiomyocytes. In addition, Ang-(1-7) decreased intracellular Ca2+ transient and sarcoplasmic reticulum Ca2+ content in PV cardiomyocytes. A779 (a Mas receptor blocker, 3 ?M), L-NAME (a NO synthesis inhibitor, 100 ?M) or wortmannin (a specific PI3K inhibitor, 10 nM) attenuated the effects of Ang-(1-7) (100 nM) on PV spontaneous electric activity. Conclusion Ang-(1-7) regulates PV electrophysiological characteristics and Ca2+ homeostasis via Mas/PI3K/eNOS signaling pathway. This article is protected by copyright. All rights reserved.