P5A- and P5B- ATPases, or collectively P5-ATPases, are eukaryotic-specific ATP-dependent transporters that are important for the function of the endoplasmic reticulum (ER) and endo-/lysosomes. However, their substrate specificities had remained enigmatic for many years. Recent cryo-electron microscopy (cryo-EM) and biochemical studies of P5-ATPases have revealed their substrate specificities and transport mechanisms, which were found to be markedly different from other members of the P-type ATPase superfamily. The P5A-ATPase extracts mistargeted or mis-inserted transmembrane helices from the ER membrane for protein quality control, while the P5B-ATPases mediate export of polyamines from late endo-/lysosomes into the cytosol. In this review, we discuss the mechanisms of their substrate recognition and transport based on the cryo-EM structures of the yeast and human P5-ATPases. We highlight how structural diversification of the transmembrane domain has enabled the P5-ATPase subfamily to adapt for transport of atypical substrates.