There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound-healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose–response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA-AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2+ oscillations in fibroblasts. Data indicate that cell Ca2+ plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways.