This study systematically explored the role of NEDD8 in pediatric acute myeloid leukemia (AML) through patient sample analysis, database mining, and in vitro experiments. Our results demonstrated that NEDD8 was significantly overexpressed in newly diagnosed pediatric AML patients and was associated with poor survival outcomes. Functional enrichment analysis of the TARGET database further revealed a strong correlation between NEDD8 and cancer-related pathways. In vitro experiments showed that NEDD8 knockdown significantly inhibited the proliferation of AML cells (THP-1 and MV4-11) and induced cell cycle arrest. Collectively, these findings highlight the critical role of NEDD8 in pediatric AML pathogenesis and suggest its potential as both a prognostic biomarker and a therapeutic target.