Polyamines, such as putrescine, spermidine and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system (PTS) using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor MGBG (methylglyoxal bis- (guanylhydrazone)), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in wild-type cells induced a CHO-MG phenotype, demonstrating a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3 as a major component of the mammalian PTS that confers sensitivity to MGBG and that has been previously genetically linked with pulmonary arterial hypertension.