Hypoxic pulmonary vasoconstriction (HPV) is a physiological response that diverts blood flow to oxygen rich areas of the lung from those areas deprived of oxygen (Sylvester et al. 2012). The AMP-activated protein kinase (AMPK, Hardie, 2007) has been proposed to underpin cardiorespiratory adjustments during hypoxia (Evans et al., 2006), and pharmacological studies support the view that AMPK mediates HPV (Evans et al., 2005). The aim of the present investigation was to determine by conditional gene deletion whether or not the LKB1-AMPK signalling pathway in pulmonary arterial myocytes is indeed necessary for HPV. We assessed the impact of single and dual deletion of the genes for AMPKα1 and α2 catalytic subunits, and also assessed the impact of deleting the genes for the upstream kinases that activate AMPK in response to metabolic stress and increases in cytoplasmic calcium, respectively, namely LKB1 and CAMKK-β. Patch-clamp electrophysiological experiments show that deletion of AMPK catalytic subunits markedly attenuates Kv current inhibition by hypoxia in pulmonary arterial myocytes. Moreover, non-invasive Echo Doppler ultrasound revealed that HPV in-vivo was significantly inhibited by deletion in arterial myocytes of LKB1 or the AMPKα1 and α2 subunits, but remained unaffected following global knockout of CAMKK-β. Our findings demonstrate that AMPK determines, at least in part, pulmonary vascular responses to acute hypoxia at the molecular, cellular and system level. We therefore conclude that the LKB1-AMPK signalling pathway is required for HPV.