Cardiovascular disease is associated with androgen hormones, particularly testosterone. Platelets play an important role in cardiovascular events as it can undergo inappropriate activation, adhesion, and aggregation, thus supporting the formation of a thrombus. The attachment of platelets to endothelial cells is influenced by CD40-CD40L interaction. Hyperglycemia can increase platelet reactivity and activate endothelial cells. To examine the influence of testosterone to the expression of CD40 in hyperglycemic endothelial cells, we did an in vitro laboratory study using post test only control group design. Study subject was the primary culture of endothelial cells derived from human umbilical vein endothelial cells (HUVEC). Independent variables were testosterone that was given in 3 doses: 1 nM, 10 nM, and 100 nM. High glucose environment was given in 22,4 mM. The expression of endothelial CD40 was measured using enzyme-linked immunosorbent assay (ELISA). This study showed that endothelial CD40 expression in high glucose medium without testosterone (12.14 ± 4.24 pg/ml) was not significantly different to those with 1 nM testosterone (19.8 pg/ml; p = 0.38), 10 nM testosterone (17.96 pg/ml; p = 0.467), and 100 nM testosterone (25.98 pg/ml; p = 0.229). Moreover using one-way ANOVA, incremental doses of testosterone did not significantly influence endothelial CD40 expression in high glucose medium (p = 0.428). In conclusion, testosterone does not influence endothelial CD40 expression in high glucose environment.