BACKGROUND: The multipotency of dental pulp stem cells (DPSCs) plays a crucial role in dental tissue regeneration, yet its regulatory mechanisms remain incompletely understood. This study aimed to investigate the role of natriuretic peptide receptor 3 (NPR3) in regulating DPSCs functions and validate the mechanism of its targeted inhibitor ligustrazine (TMP). METHODS: NPR3 expression in DPSCs was examined by Western blot and immunohistochemistry. The effects of NPR3 on DPSCs colony formation, migration, and differentiation were investigated through overexpression and knockdown strategies. The relationship between NPR3 and ERK1/2 pathway was explored using molecular biological approaches. High-throughput drug screening was employed to identify TMP as an NPR3 inhibitor, followed by mechanism validation. RESULTS: NPR3 was highly expressed in mature odontogenic DPCs, with its expression levels closely correlated with DPSCs functions. Functional assays demonstrated that NPR3 inhibited DPSCs colony formation, migration, and differentiation capabilities, while NPR3 knockdown significantly enhanced these functions. Mechanistic studies revealed that NPR3 influenced DPSCs functions through positive regulation of ERK1/2 phosphorylation. Through high-throughput screening, we identified TMP as a specific NPR3 inhibitor that promoted DPSCs functions. Rescue experiments further confirmed that NPR3 overexpression or ERK1/2 inhibitor SCH772984 attenuated TMP-induced enhancement, validating TMP's action through the NPR3/MAPK pathway. CONCLUSION: This study reveals the crucial role of the NPR3/MAPK pathway in regulating DPSCs multipotency and demonstrates that TMP enhances DPSCs functions through targeted inhibition of this pathway, providing new therapeutic strategies and drug targets for dental tissue regeneration.