Aim To investigate the relationship between acute ischemic stroke (AIS) and miR-335 gene(MIR335) promoter methylation of peripheral blood leucocyte in patients. Methods The study included 30 AIS patients and 30 age- and sex-comparable healthy controls. Bioinformatics database analysis was used to describe the CpG island of MIR335 promoter region. The quantitative methylation level in the 33 CpG sites of the MIR335 promoter was measured by bisulfite sequencing PCR in each participant. Stroke severity was evaluated by the National Institutes of Health Stroke Scale (NIHSS). Results Bioinformatic searches show that the fragment surrounding the transcription start site of MIR335/MEST exhibits a characteristic CpG island which overlaps with the promoter region. Compared with healthy controls, the levels of MIR335 promoter methylation were significantly higher in stroke patients (P<0.01). The level of MIR335 promoter methylation was negatively correlated to the level of plasma miR-335(r=－0.72,P<0.01), and positively correlated to NIHSS (r=0.74, P<0.01). Conclusion Ischemic stroke patients have higher MIR335 promoter methylation levels in the peripheral blood leucocyte than those in the controls. The study implies that hypermethylation of MIR335 promoter CpG island might be involved in ischemic stroke.