Although baseline plasma homocysteine levels are related to pemetrexed toxicities in patients treated without folate supplementation, the relationship between these parameters in patients treated with folate supplementation is not well understood. The pretreatment plasma homocysteine levels were measured in non-small-cell lung cancer patients treated with pemetrexed alone under folate supplementation. Pemetrexed (500 mg/m) was administered every 3 weeks. As folate supplementation, folic acid (0.5 mg) was orally administered daily and vitamin B12 (1 mg) was injected intramuscularly every 9 weeks starting at least 1 week before treatment. The rate of toxicities during the first cycle of pemetrexed treatment with folate supplementations was evaluated and the relationship between the plasma homocysteine levels and toxicities was examined. Between June 2009 and November 2010, 58 patients were enrolled in this study. The median pretreatment plasma homocysteine level was 7.7 μmol/ml (3.5-34.6 μmol/ml). The pretreatment plasma homocysteine levels were above 11.5 μmol/ml in nine patients (15.5%). The pretreatment plasma homocysteine level correlated significantly with the nadir of the absolute counts of leukocytes, neutrophils, and thrombocytes (r = -0.374, P = 0.004; r = -0.286, P = 0.028; r = -0.324, P = 0.012, respectively). In addition, the rates of decrease in leukocytes, neutrophils, and thrombocytes correlated significantly with the pretreatment plasma homocysteine level (r = +0.378, P = 0.003; r = +0.335, P = 0.009; r = +0.363, P = 0.005, respectively). The plasma homocysteine level is associated with hematological toxicities in patients receiving pemetrexed with folate supplementation.