We previously showed that hydrogen peroxide (H₂O₂) induced resistance artery relaxation independent of endothelium. Thus, in this study we investigated the mechanism of relaxation induced by H₂O₂ on human renal vascular smooth muscle cell (HVSMC). HVSMC were stimulated with H₂O₂ and/or angiotensin II (Ang II), proline-rich-tyrosine-kinase-2 (PYK2), ERK1/2 MAP-Kinase, and myosin light chain 20 phosphorylation (Lc20) were assessed using Western blot analysis in the presence of potassium channel blockers, MAP-Kinase, and nitric oxide synthesis (NOS) inhibitors. H₂O₂ increased PYK2 and ERK1/2 phosphorylation, and at the same time decreased Lc20 phosphorylation. AngII increased phosphorylation of PYK2, ERK1/2 and Lc20, whereas, the pretreatment of HVSMC with H₂O₂ decreased Lc20 phosphorylation induced by AngII. MEK inhibition, decreased ERK1/2 phosphorylation, but had no effect on the inhibition of phosphorylation of Lc20 induced by H₂O₂. The inhibition of Ca²⁺-dependent K⁺ channels (BKCa) and NOS did not block the decrease of Lc20 phosphorylation in response to H₂O₂. On the other hand, pretreatment of HVSMC with 60 mM of KCl, increased rather than decreased Lc20 phosphorylation in response to H₂O₂. This study shows the evidence that H₂O₂ acts as a relaxing factor and as an activator of PYK2 and ERK1/2 in Human renal VSMC. The relaxation induced by H₂O₂ is independent of BKCa, ERK1/2 MAP-Kinase and NOS pathways. The relaxing effect to H₂O₂ changes to contracting effect when the potassium channels are compromised.