GP300 is a high molecular weight glycoprotein of the bovine lungworm Dictyocaulus viviparus. The Nlinked glycans are substituted with phosphorylcholine (PC) giving it immunomodulatory potential. GP300 is highly immunogenic and its recognition by IgE antibodies is correlated with protection against infection. Herewe identified and characterized the protein backbone of GP300. Mass spectrometric analysis on purified GP300 andDNA sequencing of the corresponding gene indicated that GP300 is a thrombospondin-like protein with 7 thrombospondin domains, 6 kunitz domains and 15 putative N-glycosylation sites. Purified GP300 display protease inhibitory activity. The proteinwas located in the brushborder of the gut, but also in muscles, hypodermis and the lining of the uterus. Analysis of GP300 orthologues in Haemonchus contortus and Cooperia oncophora revealed that these proteins also contain PC-substituted N-glycans and showed immunological cross-reactive responses. These data suggest the existence in nematodes of a GP300 protein family that is characterized by PC-substituted N-linked glycans attached to a thrombospondin-like protein backbone. This finding is of particular interest considering the immunomodulatory and vaccine potential of members of the GP300 family.