Aim Sphingosine‐1‐phosphate (S1P) influences resistance vessel function and is implicated in renal pathological processes. Previous studies revealed that S1P evoked potent vasoconstriction of the pre‐glomerular microvasculature, but the underlying mechanisms remain incompletely defined. We postulated that S1P‐mediated pre‐glomerular microvascular vasoconstriction involves activation of voltage‐dependent L‐type calcium channels (L‐VDCC) and the rho/rho kinase pathway. Methods Afferent arteriolar reactivity was assessed in vitro using the blood‐perfused rat juxtamedullary nephron preparation, and diameter was measured during exposure to physiological and pharmacological agents. Results Exogenous S1P (10−9–10−5 mol L−1) evoked concentration‐dependent vasoconstriction of afferent arterioles. Superfusion with nifedipine, a L‐VDCC blocker, increased arteriolar diameter by 39 ± 18% of baseline and significantly attenuated the S1P‐induced vasoconstriction. Superfusion with the rho kinase inhibitor, Y‐27632, increased diameter by 60 ± 12% of baseline and also significantly blunted vasoconstriction by S1P. Combined nifedipine and Y‐27632 treatment significantly inhibited S1P‐induced vasoconstriction over the entire concentration range tested. In contrast, depletion of intracellular Ca2+ stores with the Ca2+‐ATPase inhibitors, thapsigargin or cyclopiazonic acid, did not alter the S1P‐mediated vasoconstrictor profile. Scavenging reactive oxygen species (ROS) or inhibition of nicotinamide adenine dinucleotide phosphate oxidase activity significantly attenuated S1P‐mediated vasoconstriction. Conclusion Exogenous S1P elicits potent vasoconstriction of rat afferent arterioles. These data also demonstrate that S1P‐mediated pre‐glomerular vasoconstriction involves activation of L‐VDCC, the rho/rho kinase pathway and ROS. Mobilization of Ca2+ from intracellular stores is not required for S1P‐mediated vasoconstriction. These studies reveal a potential role for S1P in the modulation of renal microvascular tone.