Chronic hypoxia increases resting heart rate (HR), but the underlying mechanism remains incompletely understood. We investigated the relative contributions of the sympathetic and parasympathetic nervous systems, along with potential non-autonomic mechanisms, by individual and combined pharmacological inhibition of muscarinic and/or β-adrenergic receptors. In seven healthy lowlanders, resting HR was determined at sea level (SL) and after 15–18 days of exposure to 3454 m high altitude (HA) without drug intervention (control, CONT) as well as after intravenous administration of either propranolol (PROP), or glycopyrrolate (GLYC), or PROP and GLYC in combination (PROP+GLYC). Circulating noradrenaline concentration increased from 0.9 ± 0.4 nmol l−1 at SL to 2.7 ± 1.5 nmol l−1 at HA (P = 0.03). The effect of HA on HR depended on the type of autonomic inhibition (P = 0.006). Specifically, HR was increased at HA from 64 ± 10 to 74 ± 12 beats min−1 during the CONT treatment (P = 0.007) and from 52 ± 4 to 59 ± 5 beats min−1 during the PROP treatment (P < 0.001). In contrast, HR was similar between SL and HA during the GLYC treatment (110 ± 7 and 112 ± 5 beats min−1, P = 0.28) and PROP+GLYC treatment (83 ± 5 and 85 ± 5 beats min−1, P = 0.25). Our results identify a reduction in cardiac parasympathetic activity as the primary mechanism underlying the elevated HR associated with 2 weeks of exposure to hypoxia. Unexpectedly, the sympathoactivation at HA that was evidenced by increased circulating noradrenaline concentration had little effect on HR, potentially reflecting down-regulation of cardiac β-adrenergic receptor function in chronic hypoxia. These effects of chronic hypoxia on autonomic control of the heart may concern not only HA dwellers, but also patients with disorders that are associated with hypoxaemia.