Background: Sex hormone-binding globulin (SHBG) is a protein. The synthesis of SHBG protein is encoded by SHBG gene. Protein polymorphism of SHBG can occur because of a mutation of D327N exon 8 in SHBG gene. D327N mutation causes the addition of glycosylation so that molecular weight of SHBG protein increases. Protein polymorphism of SHBG is suspected to affect the levels of SHBG, testosterone, and insulin in the blood circulation. Objective:To determine the effect of protein polymorphism of SHBG on the levels of SHBG, testosterone, and insulin in healthy adult men. Materials and Methods:Serum samples were collected from 179 healthy adult men. Electrophoresis and western blotting were performed to determine the phenotype of SHBG. All subjects were divided into two groups based on SHBG phenotype; group I (normal SHBG phenotype) n = 159 subjects and group II (variant SHBG phenotype) n = 38 subjects. Result:The levels of serum SHBG, testosterone, and insulin were measured using radioimmunoassay method. The levels of SHBG, total testosterone, and free testosterone between normal SHBG phenotype do not differ with variant SHBG phenotype (p > 0.05), but insulin levels between normal SHBG phenotype compared with variant SHBG phenotype was different (p < 0.05). Conclusion:Protein polymorphisms of SHBG was not found to affect the levels of SHBG, total testosterone, and free testosterone. Protein polymorphism of SHBG was found to affect the insulin levels in the blood serum.