ISGylation is a ubiquitin-like enzymatic cascade that transfers the small modifier ISG15 to lysine residues of protein substrates. ISGylation occurs in a three-step enzymatic cascade involving UBA7 (E1), UBE2L6 (E2), and HERC5, TRIM25 or HHARI (E3) enzymes. This mechanism regulates core cellular processes, but its role in neurodevelopmental disorders remains unclear. Here, we identified individuals with neurodevelopmental disorder phenotypes harboring biallelic UBA7 gene variants and assessed their functional effects. Truncating UBA7 variants result in loss of catalytic activity, protein stability and localization. In contrast, a missense variant drives no functional defects. Fibroblasts harboring the variant p.Lys709Serfs*45 had reduced UBA7 transcript and produced a truncated and unstable UBA7 protein. These fibroblasts were unable to induce ISGylation upon interferon beta treatment, indicating a dysfunctional ISGylation system. Together, our findings identify cellular mechanisms disrupted by UBA7 variants and lay the foundation for uncovering the role of the ISGylation system and UBA7 in neurodevelopment.