Protein C (PC) is a well-characterized anticoagulant enzyme. However, the association between PC and ischemic stroke (IS) remains controversial. The aim of the present study was to investigate whether any genetic variant in the human protein C gene (PROC) was associated with susceptibility to IS in the Chinese Han population. All exons and the 5'- and 3'-untranslated regions of PROC were initially sequenced to identify informative variants. Potential abnormal variants were analyzed in a population of 788 IS patients and 1,200 healthy controls. The analysis was stratified by stroke etiology, and the results were replicated in 262 IS patients and 288 healthy controls. Finally, functional studies were performed to evaluate the effects of the variant. A three-nucleotide duplication/deletion variant (c.574_576del) was identified and found to be significantly associated with IS (OR 2.56, 95 % CI 1.45-4.52, P = 0.001). Stratification by stroke etiology after adjustment for IS risk factors showed that this association persisted in the lacunar and cardioembolic subtypes (P < 0.001 and P = 0.008, respectively) but not in the atherothrombotic and undetermined subtypes (P = 0.070 and P = 0.998, respectively). The functional studies showed a significant difference in the anticoagulant activity of PC in c.574_576del carriers and non-carriers (P < 0.001). Our results suggested that the novel PROC c.574_576del variant is a possible genetic determinant of an increased risk of IS and diminished anticoagulant activity of PC.