Chronic graft-versus-host disease (cGVHD) is a major complication of hematopoietic stem cell transplantation. Dry eye disease is the prominent ocular sequel of cGVHD and is caused by excessive inflammation and fibrosis in the lacrimal glands. Heavy chain-Hyaluronan/Pentraxin 3 (HC-HA/PTX3) is a complex purified from human amniotic membrane (AM) and known to exert anti-inflammatory and anti-scarring actions. In this study, we utilized a mouse model of cGVHD to examine whether HC-HA/PTX3 could attenuate dry eye disease elicited by cGVHD. Our results indicated that subconjunctival and subcutaneous injection of HC-HA/PTX3 preserved tear secretion and conjunctival goblet cell density and mitigated inflammation and scarring of the conjunctiva. Such therapeutic benefits were associated with suppression of scarring and infiltration of inflammatory/immune cells in the lacrimal glands. Furthermore, HC-HA/PTX3 significantly reduced the extent of infiltration of CD45+ CD4+ IL-17+ cells, CD45+CD34+ collagen I+ CXCR4+ fibrocytes, and HSP47+ activated fibroblasts that were accompanied by upregulation of collagen type Iα1, collagen type IIIα1 and NF-kB in lacrimal glands. Collectively, these pre-clinical data help prove the concept that subcutaneous and subconjunctival injection of HC-HA/PTX3 is a novel approach to prevent dry eye disease caused by cGVHD and allow us to test its safety and efficacy in future human clinical trials.