To determine whether TGF-βs and the effect on endothelial cell migration were altered during fresh frozen plasma (FFP) refrigeration, ELISA assay was carried out to quantify TGF-βs protein levels in FFP stored at 4℃ for up to 5 days. Human pulmonary microvascular endothelial cells (HPMECs) were treated with various concentrations of Day 0 and Day 5 FFPs or 10% Lactated Ringer's (LR) and were subjected to migration assay or Western blot analysis. ALK5 siRNA or ALK5 inhibitor were used to block FFP-induced Smad3 signaling in EC cells. It was found that TGF-β1 protein levels increased in a time-dependent fashion at a rate of 244.31 ng/(L·d) (P < 0.05) and greater activation of its downstream mediators Smad2/3 during storage of FFP (P < 0.05). Both Day 0 FFP and Day 5 FFP stimulated EC migration in vitro; however, the effect of Day 5 FFP was significantly reduced. Inhibition of TGF-β typeⅠ receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of Day 5 FFP on EC migration to a comparable level as Day 0 FFP. These data suggest that the increased TGF-β levels during FFP storage contributes to the deterioration of stored FFP's effects on EC migration. A novel molecular mechanism contributing to the reduced efficacy of stored FFP was identified.