High-density lipoprotein (HDL) cholesterol levels are associated with a decreased risk of coronary artery disease. Several genome-wide association studies that have examined HDL cholesterol levels have implicated myosin light chain 2 regulatory cardiac slow (MYL2) as a possible causal factor. Herein, the association between the rs3782889 single-nucleotide polymorphism (SNP) in the MYL2 gene and HDL cholesterol levels was tested in the Korean population. A total of 4294 individuals were included in a replication study with MYL2 SNP rs3782889. SNP rs3782889 in the MYL2 gene was associated with mean HDL cholesterol level (effect per allele, -1.055 mg dl(-1), P=0.0005). Subjects with the CT/CC genotype had a 1.43-fold (range 1.19-1.73-fold) higher risk of an abnormal HDL cholesterol level (<40 mg dl(-1)) than subjects with the TT genotype. When analyzed by sex, the MYL2 association was stronger in men than that in women. When analyzed by body mass index (BMI), the MYL2 association was much stronger in male subjects with BMI ⩾26.44 kg m(-2) (odds ratio (OR)=2.68; 95% confidence interval (CI)=1.87-3.84; P<0.0001) than that in male subjects with BMI <26.44 kg m(-2). When compared with subjects having the TT genotype and BMI <26.44 kg m(-2), ORs (95% CI) were 3.30 (2.41-4.50) in subjects having the CT/CC genotype and BMI ⩾26.44 kg m(-2) (P for interaction <0.0001). Our results clearly demonstrate that genetic variants in MYL2 influence HDL cholesterol levels in Korean obese male subjects.