It is a common clinical phenomenon that blood infiltrates into the injured tendon caused by sports injuries, accidental injuries, and surgery. However, the role of blood infiltration into the injured tendon has not been investigated. We established a rat model in which the injured Achilles tendon was infiltrated with autologous whole blood and confirmed that blood caused acute inflammation in the short term and more severe heterotopic ossification (HO) in the long term. Then we found that blood treatment increased cell apoptosis and decreased cell adhesion and tenogenic gene expression of TSPCs. Furthermore, Blood treatment promoted osteochondrogenic differentiation of TSPCs. Next, we used RNA-seq to find that the PI3K/AKT signaling pathway was activated in blood-treated tendon tissues. By inhibiting PI3K with a small molecule drug LY294002, the expression of osteochondrogenic genes was markedly downregulated while the expression of tenogenic genes was significantly upregulated. Our findings indicate that the upregulated PI3K/AKT signaling pathway is implicated in the aggravation of tendon HO. Therefore, inhibitors targeting the PI3K/AKT pathway would be a promising approach to treat blood-induced tendon HO.