Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.