L-Arginine transport is mediated by the cationic/neutral amino acid transport system y+L and cationic amino acid transporters y+/CATs in human umbilical vein endothelial cells (HUVECs). System y+/CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y+L involvement in NO synthesis in endothelium. We investigated the role of system y+L in NO synthesis in HUVECs. Transport of 1.5 μM L-arginine was inhibited (P < 0.05) by L-lysine (Ki, 1.4 μM), L-leucine (Ki, 1.8 μM) and L-phenylalanine (Ki, 4.1 μM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y+/CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 μM L-arginine transport, but inhibited (92 ± 3%) 100 μM L-arginine transport. L-Arginine transport in the presence of NEM was saturable (Vmax, 0.37 ± 0.02 pmol (μg protein)−1 min−1; Km, 1.5 ± 0.3 μM) and competitively inhibited by L-leucine in the presence of Na+ (Vmax, 0.49 ± 0.06 pmol (μg protein)−1 min−1; Km, 6.5 ± 0.9 μM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y+L. NG-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 μM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (Vmax, 0.15 ± 0.008 pmol (μg protein)−1 min−1; Km, 1.4 ± 0.3 μM) and NO synthesis. However, 25 mM D-glucose increased NO synthesis and L-arginine transport via system y+. Thus, L-arginine transport through system y+L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine-NO pathway is altered, such as in diabetes mellitus.