Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO₂). The effect of LNO₂ on human platelets was examined to define how nitrated lipids might behave <i>in vivo</i>. LNO₂, but not LA or 3-nitrotyrosine, dose dependently (0.5–10 μm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO₂ caused slight mobilization of calcium (Ca²⁺) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca²⁺ elevations. LNO₂ did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1<i>H</i>-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO₂treatment of platelets, and the adenylyl cyclase inhibitors 2′,5′-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO₂significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO₂. The platelet inhibitory actions of LNO₂indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.