Updated project metadata. Polyamines, including putrescine (PUT), spermidine (SPD) and spermine (SPM), are abundant polycations supporting various cellular functions. Their cellular content is tightly controlled by biosynthesis, degradation and uptake or export via the polyamine transport system (PTS). However, the mammalian PTS remains poorly characterized. Mutated Chinese hamster ovary cells, CHO-MG, are frequently used to study the PTS owing to their phenotype of reduced polyamine uptake and resistance to methylglyoxal bis-(guanylhydrazone) (MGBG), a toxic polyamine biosynthesis inhibitor. Yet, the underlying genetic defect remains enigmatic. We recently proved that the P5B-type ATPase, ATP13A2, is a polyamine transporter and here, we analyzed whether the P5B-type ATPases are deficient in CHO-MG. Label-free shogun proteomics found that ATP13A3 expression is reduced in CHO-MG. Mutations in the ATP13A3 gene were found to critically disturb the protein sequence. Interestingly, depleting ATP13A3 in the wild-type CHO cells induced a CHO-MG phenotype, whereas its complementation in CHO-MG rescued the phenotype. Together, we demonstrate that defective ATP13A3 contributes to the CHO-MG phenotype, designating ATP13A3 as a new member of the mammalian PTS that may partially overlap in function with ATP13A2.