In the yeastSaccharomyces cerevisiae, the Nem1–Spo7 complex is a protein phosphatase that activates Pah1 phosphatidate phosphatase at the nuclear–endoplasmic reticulum membrane for the synthesis of triacylglycerol. The Nem1–Spo7/Pah1 phosphatase cascade largely controls whether phosphatidate is partitioned into the storage lipid triacylglycerol or into membrane phospholipids. The regulated synthesis of the lipids is crucial for diverse physiological processes during cell growth. Spo7 in the protein phosphatase complex is required as a regulatory subunit for the Nem1 catalytic subunit to dephosphorylate Pah1. The regulatory subunit contains three conserved homology regions (CR1, CR2, and CR3). Previous work showed that the hydrophobicity of LLI (residues 54–56) within CR1 is important for Spo7 function in the Nem1–Spo7/Pah1 phosphatase cascade. In this work, by deletion and site-specific mutational analyses, we revealed that CR2 and CR3 are also required for Spo7 function. Mutations in any one of the conserved regions were sufficient to disrupt the function of the Nem1–Spo7 complex. We determined that the uncharged hydrophilicity of STN (residues 141–143) within CR2 was required for Nem1–Spo7 complex formation. In addition, the hydrophobicity of LL (residues 217 and 219) within CR3 was important for Spo7 stability, which indirectly affected complex formation. Finally, we showed the loss of Spo7 CR2 or CR3 function by the phenotypes (e.g., reduced amounts of triacylglycerol and lipid droplets, temperature sensitivity) that are attributed to defects in membrane translocation and dephosphorylation of Pah1 by the Nem1–Spo7 complex. These findings advance knowledge of the Nem1–Spo7 complex and its role in lipid synthesis regulation.