BACKGROUND: Variants of Ectodysplasin A1 (EDA1) regulate the proliferation, migration, and odontogenic differentiation of human dental pulp stem cells (hDPSCs). Further study of these variants could reveal the mechanism by which EDA1 induces tooth development. METHODS: The following groups of hDPSCs were studied: those expressing wild-type (Wt) EDA1, those expressing EDA1 non-syndromic tooth agenesis (NSTA) variants (NSTA-A259E, NSTA-S374R), those expressing a syndrome type (STA) variant of EDA1 (STA-H252L), and those transformed with the empty vector (NC, negative control). hDPSCs proliferation was assessed using Cell Counting kit 8 assays. Flow cytometry was employed to assess hDPSCs cell cycle distribution. Transwell and wound-healing assays were employed to assess hDPSCs migration. hDPSCs mineralization was induced using odontogenic differentiation medium. RNA sequencing of the various hDPSCs groups was carried out to identify enriched pathways and hub genes. Hub gene expression was confirmed using quantitative realtime reverse transcription PCR (qRT-PCR). RESULTS: Wt-EDA1 promoted hDPSCs proliferation and G0/G1 to S transition significantly compared with the NSTA-EDA1 and STA-EDA1 groups (p < 0.01). The NSTA-EDA1 and STA-EDA1 groups did not show significant differences between them (p > 0.05). Relative to that in the NSTA-EDA1 and STA-EDA1 groups, Wt-EDA1 enhanced hDPSCs migration (p < 0.01). According to alkaline phosphatase and Alizarin Red staining, compared to the Wt-EDA1 group, hDPSCs odontogenic differentiation was inhibited and proliferation was ablated in the NSTA-EDA1 and STA-EDA1 groups (p < 0.01). RNA sequencing showed enrichment of the MAPK signaling and osteoclast differentiation pathways, identifying FOS and JUN as differentially expressed hub genes. qRT-PCR demonstrated that, unlike the Wt-EDA1 group, the EDA1 variant groups could not promote FOS mRNA expression. CONCLUSIONS: In hDPSCs, EDA1 variants could not promote FOS expression, which inhibited hDPSCs odontogenic differentiation and ablated their proliferation.