Few studies have linked homocysteine, B vitamins and/or genetic defects to the risk of hypertension. The purpose of this study was to investigate homocysteine, B-vitamins, and genetic mutation in relation to the risk of hypertension. Subjects were assigned to the hypertension (HTN) group (n = 50) or non-hypertension (non-HTN) group (n = 123). All subjects' blood pressure (systolic blood pressure, SBP; diastolic blood pressure, DBP), biochemical values, plasma homocysteine, pyridoxal 5'-phosphate (PLP), serum folate, vitamin B12 concentrations, and methylenetetrafolate reductase (MTHFR) 677C-->T gene polymorphism were measured. Results showed that subjects with T-allele were positively associated with DBP (beta = 4.22, p = 0.04) but the significance became weaker (p = 0.06) after homocysteine and B vitamins were additionally adjusted. A significant association of plasma PLP with SBP remained (beta = -0.06, p = 0.01) even after homocysteine and T-allele genotypes were additionally adjusted (beta = -0.07, p = 0.02). The combined presence of low PLP (< 30 nmol/L) and carried T-allele enhanced the risk of hypertension and the risk magnitude was substantially greater (OR, 16.44, p < 0.001). Taken together, the results show that low plasma PLP levels and MTHFR 677C-->T genotypes might be significant risk factors for hypertension.