Platelets are anucleate cells whose dysregulation contributes to thrombocytopenia during sepsis. Thrombocytopenia is an early complication of Gram-negative infection, in which lipopolysaccharide (LPS) serves as a principal mediator; however, its precise contribution remains unclear. In this study, LPS, at concentration 10 µg/mL, did not induce human platelet aggregation but significantly potentiated low-dose collagen (0.5 μg/mL)-induced aggregation, ATP release, intracellular calcium levels ([Ca²⁺]i) and P-selectin expression. Scanning electron microscopy revealed that either collagen or LPS activated filopodia elongation in human platelets, whereas LPS combined with collagen further activated the phenotype of platelet activation (lamellipodia formation). Beyond these activation responses, LPS also increased TLR4 expression and triggered hallmark apoptotic events, including mitochondrial depolarization, Bax expression, caspase-8 and caspase-3 activation, and phosphatidylserine exposure, concomitant with downregulation of Bcl-2. Moreover, LPS-induced apoptotic platelets displayed ultrastructural changes, characterized by membrane blebbing and filopodia loss. Thus, these findings present the first evidence that LPS enhances platelet aggregation in association with apoptosis through the TLR4–Bax/Bcl-2–mitochondrial dysfunction–caspase-8/3 activation signaling pathway, providing mechanistic insight into sepsis-associated thrombocytopenia.