Background: Neurodevelopmental disorders (NDDs) are a group of diseases that severely affect the physical and mental health of children. The PPP2R5D gene encodes B56δ, the regulatory subunit of protein phosphatase 2A (PP2A). NDDs related to the PPP2R5D gene have recently been defined as Houge–Janssens syndrome 1. Methods: Clinical/whole exome sequencing was performed on approximately 3000 patients with NDDs from 2017 to 2023. In vitro experiments were performed to assess the impairment of variants to protein expression and the assembly of PP2A holoenzyme. The genetic information and phenotypes of the reported patients, as well as patients in this study, were summarized, and the genotype–phenotype relationship was analyzed. The probability of pathogenic missense variants in PPP2R5D was predicted using AlphaMissense (AM), and the relationship between certain phenotype and 3D protein structural features were analyzed. Results: Thirteen new patients carrying twelve PPP2R5D gene variants were detected, including five novel missense variants and one novel frameshift variant. In vitro experiments revealed that the frameshift variant p.H463Mfs*3 resulted in a ~50 kDa truncated protein with lower expression level. Except for E420K and T536R, other missense variants impaired holoenzyme assembly. Furthermore, we found that pathogenic/likely pathogenic (P/LP) variants that have been reported so far were all missense variants and clustered in three conserved regions, and the likelihood of P/LP mutations located in these conserved regions was extremely high. In addition, the macrocephaly phenotype was related to negatively charged residues involved in substrate recruitment. Conclusions: We reported thirteen new patients with PPP2R5D gene variants and expanded the PPP2R5D variant spectrum. We confirmed the pathogenicity of novel variants through in vitro experiments. Our findings in genotype–phenotype relationship provide inspiration for genetic counseling and interpretation of variants. We also provide directions for further research on the mechanism of macrocephaly phenotype.