Objective To determine whether concentrations of plasma homocysteine and status of folate and vitamin B12 were altered in patients with heroin dependence compared with control subjects. Methods We recruited 20 patients with heroin addiction. Twenty age-and sex-matched healthy volunteers as the control group were enrolled. Blood concentrations of homocysteine, folate and vitamin B12 were measured in a fasted status. Results Mean homocysteine level was higher in the heroindependence group than in the control group [18.3 ± 4.5 (Q1 = 14.7, Q2 = 17.6, Q3 = 19.2) vs. 8.4 ± 2.7 (Q1 = 5.9, Q2 = 8.5, Q3 = 10.5) μmol/L, P < 0.001]. Patients in heroinaddictiongroup had signi cantly lower folate levels compared with the control group [6.6 ± 2.6 (Q1=4.6, Q2=6.4, Q3=8.1) vs. 12.7 ± 2.5 (Q1 = 11.1, Q2 = 13.0, Q3 = 17.5) ng/mL, P < 0.001]. There was significant difference on plasma B12 concentrations between heroin users and controls [384.1 ± 228.8 (Q1=312.0, Q2=348.5, Q3=374.5) vs. 399.4 ± 45.1 (Q1=358.0, Q2=394.5, Q3=428.3) Pg/mL, P < 0.001]. Heroin users group demonstrated a negative correlation of homocysteine with folate , which was statistically significant in heroin users groups (r = - 0.614; P = 0.004), there was a trend towards a negative correlation of homocysteine with B12 in heroin users (r = -0.605; P = 0.005), respectively. Conclusions We conclude that patients with heroin dependence are prone to high concentration of plasma homocysteine.