Acylated homoserine lactones (AHL) such as N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C HSL) and N-butyryl-l-homoserine lactone (C HSL) are the most common autoinducer molecules in Pseudomonas aeruginosa. These AHL molecules not only regulate the expression of virulence factors but also have been shown to interfere with the host cell and modulate its functions. Recently, we reported that 3-oxo-C HSL but not C HSL causes cytosolic Ca rise and ROS production in platelets. In this study, we examined the potential of AHLs to induce apoptosis in the human blood platelet. Our result showed that 3-oxo-C HSL but not C HSL causes phosphatidylserine (PS) exposure, mitochondrial dysfunction (mitochondrial transmembrane potential loss, and mitochondrial permeability transition pore (mPTP) formation). Besides, 3-oxo-C HSL also inhibited thrombin-induced platelet aggregation and clot retraction. The pretreatment of an intracellular calcium chelator BAPTA-AM or ROS inhibitor (DPI) significantly attenuated the 3-oxo-C HSL induced apoptotic characters such as PS exposure and mitochondrial dysfunctions. These data, including our previous findings, confirmed that 3-oxo-C HSL induced intracellular Ca mediated ROS production results in the activation and subsequent induction of apoptotic features in platelets. Our results demonstrated that the 3-oxo-C HSL modulates the functions of platelets that may cause severe thrombotic complications in P. aeruginosa infected individuals.