Nicotine is the primary addictive component of tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. To assess the underlying mechanisms, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days before performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the first to third thoracic vertebrae, and β-adrenergic responsiveness was additionally evaluated following norepinephrine (NE) perfusion. Baseline ex vivo heart rate (HR) and SNS stimulation threshold were higher in NIC versus CT ( = 0.004 and = 0.003, respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC versus CT at baseline ( = 0.002) and during SNS ( = 0.0003), with similar results obtained for Ca transient alternans. SNS shortened the PCL at which alternans emerged in CT but not in NIC hearts. NIC-exposed hearts tended to have slower and reduced HR responses to NE perfusion, but ventricular responses to NE were comparable between groups. Although fibrosis was unaltered, NIC hearts had lower sympathetic nerve density ( = 0.03) but no difference in NE content versus CT. These results suggest both sympathetic hypoinnervation of the myocardium and regional differences in β-adrenergic responsiveness with NIC. This autonomic remodeling may contribute to the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with long-term use. Here, we show that chronic nicotine exposure was associated with increased heart rate, increased susceptibility to alternans, and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to sympathetic hypoinnervation of the myocardium and diminished β-adrenergic responsiveness of the sinoatrial node following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this proarrhythmic remodeling.