Hydrogen sulfide (H₂S) is one of three endogenous gases, along with carbon monoxide (CO) and nitric oxide (NO), that exerts a variety of important vascular actions in vivo. Although it has been demonstrated that CO or NO can trigger the development of a preconditioned phenotype in postischemic tissues, it is unclear whether H₂S may also induce protection in organs subsequently exposed to ischemia/reperfusion (I/R). Thus, we postulated that preconditioning with an exogenous H2S donor (NaHS-PC) would inhibit leukocyte rolling (LR) and adhesion (LA) induced by subsequent exposure to I/R. We used intravital microscopic techniques to demonstrate that NaHS-PC 24 hrs, but not 1 hr, prior to I/R causes postcapillary venules to shift to an anti-inflammatory phenotype in wild-type (WT) mice such that these vessels failed to support LR and LA during reperfusion. The protective effect of NaHS-PC on LR was largely abolished by coincident pharmacologic inhibition of NO synthase in WT animals and was absent in eNOS-deficient mice. A similar pattern of response was noted in WT mice treated concomitantly with NaHS plus p38 mitogen activated protein kinase (MAPK) inhibitors (SB203580 or SK86002). While the reduction in LA induced by antecedent NaHS was attenuated by pharmacologic inhibition of NOS or p38 MAPK in WT mice, the anti-adhesive effect of NaHS was still evident in eNOS-/- mice. Thus, NaHS-PC prevents LR and LA by triggering the activation of an eNOS- and p38 MAPK-dependent mechanism. However, the role of eNOS in the anti-adhesive effect of NaHS-PC was less prominent than its effect to reduce LR.