Hypohidrotic Ectodermal Dysplasia (HED) is a rare genetic disorder characterized by hypodontia, hypohidrosis, and hypotrichosis. The study aims to identify a novel mutation in the EDA2R gene in a 20-year-old female with HED and investigate its impact on the NF-κB signaling pathway. Whole genome sequencing confirmed the mutation, and bioinformatic tools predicted it to be pathogenic by destabilizing the EDA2R structure and weakening its interaction with EDA-A2. Molecular dynamics simulation and binding free energy calculations further revealed reduced hydrogen bond formation in the mutant EDA2R/EDA-A2 complex, while molecular docking and AlphaFold analyses indicated decreased binding to TRAF3 and TRAF6. In vitro experiments demonstrated that cells expressing the mutant EDA2R had significantly reduced proliferation and NF-κB activity, along with impaired nuclear translocation of NF-κB p65. However, Western blot analysis showed that the JNK signaling pathway remained unaffected. This study identifies a novel missense mutation in EDA2R and introduces a new pathogenic mechanism of HED, emphasizing the crucial role of EDA2R in regulating NF-κB signaling.