Sphingomyelin phosphodiesterase 1 (SMPD1) converts sphingomyelin into ceramide and phosphocholine; hence, loss of SMPD1 function causes abnormal accumulation of sphingomyelin in lysosomes, which results in the lipid-storage disorder Niemann-Pick disease (types A and B). SMPD1 activity is dependent on zinc, which is coordinated at the active site of the enzyme, and although SMPD1 has been suggested to acquire zinc at the sites where the enzyme is localized, precisely how SMPD1 acquires zinc remains to be clarified. Here, we addressed this using a gene-disruption/reexpression strategy. Our results revealed that Zn transporter 5 (ZNT5)-ZNT6 heterodimers and ZNT7 homodimers, which localize in the compartments of the early secretory pathway, play essential roles in SMPD1 activation. Both ZNT complexes contribute to cellular sphingolipid metabolism by activating SMPD1 because cells lacking the functions of the two complexes exhibited a reduced ceramide to sphingomyelin content ratio in terms of their dominant molecular species and an increase in the sphingomyelin content in terms of three minor species. Moreover, mutant cells contained multilamellar body-like structures, indicative of membrane stacking and accumulation, in the cytoplasm. These findings provide novel insights into the molecular mechanism underlying the activation of SMPD1, a key enzyme in sphingolipid metabolism.