Changes in diurnal rhythmicity in blood pressure (BP) are associated with hypertension and consequent cardiovascular damage. The involvement of diurnal rhythmicity as a pathogenic factor in hypertension is not fully understood. Since the hormone melatonin (MLT) regulates circadian rhythm, it was also of interest to determine whether this hormone played a role in hypertension-related alterations in circadian rhythm. Thus the aim of this study was to examine the mechanisms underlying MLT-mediated antihypertension. Human umbilical vein endothelial cells were incubated with MLT under 25 kPa pressure to simulate hypertension. Vasoactive substances including endothelin (ET), angiotensin II (Ang II), nitric oxide (NO), and endothelial nitric oxide synthase (eNOS) were measured using ELISA assays. Results showed that MLT produced a significant decrease in ET at 18 and 24 h and Ang II at 18 h after treatment. In contrast, MLT significantly elevated NO levels and eNOS activity at 6, 12, 18, and 24 h, indicating that these oxidant indicators may be more sensitive to MLT-induced actions. Gene chip analysis identified 121 upregulated and 214 downregulated genes at 6 h after MLT treatment, predominantly involved in DNA replication, cell cycle regulation, amino acid metabolism, and cell cycle pathway. At 18 h, 63 upregulated and 94 downregulated genes involved in circadian entrainment, cGMP-PKG signaling pathway involved in NO synthesis, as well as secretion of renin and insulin, which are associated with BP regulation. Data suggest that the circadian antihypertensive effects of MLT might be associated with decrease in ET and Ang II, accompanied by rise in NO and eNOS and that NO and eNOS appear to be early bioindicators of hormonal effect.