This study investigated whether the anthocyanin cyanidin-3-O-β-glucoside (Cy-3-g) could affect platelet apoptosis and proplatelet formation in vitro. Thrombin-stimulated or resting human platelets and Meg-01 megakaryocytes were incubated with Cy-3-g (0, 0.5, 5, or 50 μM). We found that the percentage of the platelet mitochondrial membrane potential treated with 5 and 50 μM Cy-3-g was significantly higher than control (15.50% ± 3.24% and 29.77% ± 4.06% versus 2.76% ± 1.33%, respectively; P < 0.05). Treatment with 5 and 50 μM Cy-3-g significantly increased phosphatidylserine exposure compared with control (40.56% ± 10.53% and 76.62% ± 8.28% versus 15.43% ± 3.93%, respectively; P < 0.05). Moreover, Cy-3-g significantly increased the expression of Bax, Bak, and cytochrome c while markedly decreasing Bcl-xL and Bcl-2 expression as well as stimulating caspase-3, caspase-9, caspase-8, Bid, and gelsolin cleavage in thrombin-activated platelets in a dose-dependent manner (P < 0.05). However, no significant differences were observed in the apoptosis of resting platelets when treated with Cy-3-g (P > 0.05). Furthermore, Cy-3-g significantly (P < 0.05) enhanced cell viability (50 μM versus control, 1.34 ± 0.01 versus 0.35 ± 0.02), the number of colony-forming unit-megakaryocytes (50 μM versus control, 38 ± 3 versus 8 ± 3), CD41 expression (50 μM versus control, 96.80% ± 2.55% versus 25.57% ± 2.86%), DNA ploidy (16N) (50 μM versus control, 19.73% ± 2.34% versus 4.42% ± 1.96%), and proplatelet formation (50 μM versus control, 27.5% ± 3.77% versus 7.67% ± 2.25%) in Meg-01 cells. In conclusion, Cy-3-g promotes activated platelet apoptosis and enhances megakaryocyte proliferation, differentiation, and proplatelet formation in vitro.