Abstract: Objective: To investigate the mechanisms of NAT10 in central post-stroke pain (CPSP) following thalamic hemorrhage. Methods: Part I: Utilizing single nucleus RNA sequencing (snRNA-seq) data from our research group, we conducted expression variation and cell interaction analysis related to NAT10, gene set enrichment analysis (GSEA), and construction of microRNA networks. Part II: Thirty male C57BL/6J mice, aged 7-8 weeks and weighing 25-30 g, were randomly assigned into two groups (n=15 each) using a random number table: the Control group and the CPSP group. A CPSP model was prepared by stereotactic injection of 0.01U/10nl type IV collagenase into the right ventral posteromedial and ventral posterolateral nuclei of the thalamus. Mechanical paw withdrawal frequency (PWF), thermal paw withdrawal latency (TWL), and cold paw withdrawal latency (CWL) were measured at 1 day before model preparation and 1, 3, 7, and 14 days post-modeling. After behavioral observations, the mice were sacrificed for tissue collection. NAT10 expression in thalamic tissue was detected using Western blot and immunofluorescence dual staining, along with co-localization with NeuN, GFAP, Iba1 and CD68. Results: The snRNA-seq revealed a significant upregulation of NAT10 in the CPSP model, co-expressed with the hemorrhage progression gene Col4a1, and closely related to the level of immune cell infiltration. The enriched pathways included cAMP, cGMP-PKG, Cholinergic synapse, and metabolism-related pathways, and 557 microRNAs potentially involved in the regulation of NAT10 expression were identified. In animal experiments, compared to the Control group, the PWF of the CPSP group significantly increased (P0.01), and TWL and CWL significantly shortened (P0.01) on the 1st, 3rd, 7th, and 14th days post-surgery. The protein expression level of NAT10 in the hemorrhagic thalamic tissue was significantly upregulated at 1, 3, 7, and 14 days post-modeling (P0.01); immunofluorescence results showed that NAT10 mainly overlapped with the fluorescence signal of NeuN. Conclusion: NAT10 may play an important role in the occurrence and development of CPSP following thalamic hemorrhage by affecting neuronal function.