This study was aimed to investigate the relationship between the methylation status of serotonin transporter (5-HTT) and major depressive disorder (MDD) in Chinese Han population. A total of 96 patients with MDD and 55 healthy volunteers were recruited, and the methylation index (MtI) at six positions in the cytosine-phosphate-guanosine island of 5-HTT gene was measured for each subject using bisulfite pyrosequencing. MtIs at positions 5 and 6 were higher in patients with MDD than those in controls. According to the multivariable logistic regression analysis, MtIs at positions 4 and 5 were significantly associated with MDD. Besides, depression education was an independent risk factor, whereas higher educational levels were protective factors for MDD. In addition, positions 1 and 4 were negatively correlated with weight and diurnal variation. Therefore, 5-HTT methylation might be closely related with MDD in Chinese Han population because of the correlation with diurnal variation and weight.