Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species (ROS) by different cell types contributes to sepsis. Platelets generate ROS, but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide (LPS)-treated rats to investigate the p47 phox activation and ROS generation, and its modulation by c-Src family kinase (c-Src), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) and protein kinase G (PKG). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre-incubated with different inhibitors and subsequently activated or not with ADP. Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47 phox phosphorylation and ROS generation compared with the control group (P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non-selective SFKs inhibitor PP2, the PI3K inhibitor wortmannin or the Akt inhibitor PPI-1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group (P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ, the PKG inhibitor Rp-8-Br and the PKC inhibitor GF109203X abrogated the increased p47 phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP-PKG and PKC-p47 phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.