Introduction: The sinoatrial node (SAN) is the primary pacemaker of the heart. Clinical evidence shows that SAN function differs between sexes. Males have slower resting (extrinsic and intrinsic) heart rates, and longer corrected SAN recovery time compared with females. Hypothesis: Sex based differences may be due to SAN ion channel differences. Therefore, we investigated functional behaviour and molecular makeup of SANs in male and female rats. Methods: Intracellular action potential recordings were obtained using sharp microelectrode technique (n=5 each). Gene expression was measured with quantitative PCR (n=6 each). Results: The amplitude of the action potential was significantly higher in male SANs (45.3 ± 2.4) compared with female (31.7 ± 2.5). The upstroke velocity (dV/dtmax) was significantly faster in female SANs (16.4 ± 1.6) compared with male (9.1 ± 1.5). Out of 96 transcripts investigated, two were significantly different between the sexes. L-Type Ca2+ channel, Cav1.3, and acetylcholine activated K+ channel, Kir3.1, mRNA levels were significantly higher in female SANs (66.6% and 45.6% respectively) compared with male. Immunolabelling for Cav1.3 showed levels were significantly higher in female SANs compared with male (18.6%, n=6 each). Inhibition of Cav1.3 with nifedipine (5μM) showed greater effect on the beating rate of male SANs compared with female (43%). A non-coding RNA, miR-139-3p (predicted by Ingenuity IPA software to affect Cav1.3) showed greater effect on the beating rate of male SAN preparations compared with female. Mathematical modelling showed that a higher density of Cav1.3 (as in the case of females) has a protective role from sinoatrial node arrhythmias. Conclusion: This study identified functional and molecular differences in key pacemaker ion channels in SAN between sexes. These differences advance our understanding of gender differences in cardiac electrophysiology and may have implications for gender specific design of anti-arrhythmic drugs and biological pacemakers.