Introduction: Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. It is characterized by paroxysmal abnormal ocular movements, dystonia, alternating hemiplegia, and psychomotor developmental delay. Some AHC patients associate with epilepsy. ATP1A3 was recently identified as the causative gene of AHC. Here we report the first genetic study of AHC in Chinese cohort. Methods: Fifty-two Chinese patients with AHC were recruited in this study. Genomic DNA was extracted from peripheral blood samples. Mutations of ATP1A3 were analyzed using PCR amplification and DNA sequencing. Results: ATP1A3 mutations were detected in 95.8% of typical AHC patients. At least 91.1% were de novo. Four atypical AHC patients of late onset were also mutation-positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 12 novel missense mutations (T370N, G706R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G938R) were identified in our study. Genotype-phenotype correlation analysis showed that patients with quadriplegia were more likely to carry D801N and less likely to carry E815K mutation, whereas patients with epilepsy were more likely to carry E815K mutation. Forty-two patients were treated with Flunarizine. 29 (69%) of whom showed reduced severity, duration, or frequency of hemiplegic attacks. We found no correlation between treatment effects and the three mutation hotpots (D801N, E815K and G947R).Conclusions: ATP1A3 is also the major pathogenic gene of AHC in Chinese patients. Most mutations are de novo. Some novel mutations have been found in Chinese patients. Mutation E815K is correlated with the phenotype of epilepsy.