BACKGROUND AND PURPOSE-: Hyperlipidemia is associated with platelet hyperreactivity. We hypothesized that L-4F, an apolipoprotein A-I mimetic peptide, would inhibit platelet aggregation in hyperlipidemic mice. METHODS AND RESULTS-: Injecting L-4F into apolipoprotein E (apoE)-null and low-density lipoprotein receptor-null mice resulted in a significant reduction in platelet aggregation in response to agonists; however, there was no reduction in platelet aggregation after injection of L-4F into wild-type (WT) mice. Consistent with these results, injection of L-4F into apoE-null mice prolonged bleeding time; the same result was not found in WT mice. Incubating L-4F in vitro with apoE-null platelet-rich plasma also resulted in decreased platelet aggregation. However, incubating washed platelets from either apoE-null or WT mice with L-4F did not alter aggregation. Compared with WT mice, unstimulated platelets from apoE-null mice contained significantly more 12-hydroxy 5,8,10,14- eicosatetraenoic acid, thromboxane A2, and prostaglandins D2 and E2. In response to agonists, platelets from L-4F-treated apoE-null mice formed significantly less thromboxane A2, prostaglandins D2 and E2, and 12-hydroxy 5,8,10,14-eicosatetraenoic acid. CONCLUSION-: By binding plasma-oxidized lipids that cause platelet hyperreactivity in hyperlipidemic mice, L-4F decreases platelet aggregation. © 2010 American Heart Association, Inc.