Abstract Glucose has been found to impair the inhibition of platelets with aspirin and alter the basal activity of nitric oxide synthase (NOS) in platelets. The aim of this work was to study the effects of glucose on the inhibitory pathways in activated platelets. A short-term incubation of glucose impaired the inhibition of platelet aggregation induced by agents activating an NOS-dependent pathway, such as l-arginine, adenosine and α-tocopherol. However, glucose had no effect on the inhibition induced by iloprost and BW245C, agents that activate the cyclic adenosine monophosphate (cAMP) signaling pathway. Potassium lactate attenuated the effects of the same inhibitors as glucose did. The inhibitors of glucose transport prevented the effect of glucose. Dichloroacetate, known to prevent the conversion of pyruvate to lactate and to decrease lactate in platelets, significantly attenuated the effect of glucose in platelets. The data support the suggestion that the effect of glucose on the inhibition of platelets by agents activating an NOS-dependent pathway is mediated by glucose metabolite lactate.