P-glycoprotein (P-gp), as an ATP-binding cassette transporter, transports a wide variety of substrates varying from small molecules like steroids to large polypeptides across the cell membrane in human and animals, even in aquatic animals. Although P-gp protein has attracted much attention of research, its effect on the toxicity of environmental toxicants such as antifouling biocides is still poorly understood. The goal of this study is to evaluate whether copper pyrithione (CuPT), Sea-Nine 211, dichlofluanid and tolylfluanid, four widely used antifouling agents, can be transported by P-gp in embryos of sea urchin Strongylocentrotus intermedius in the presence and absence of the P-gp inhibitor verapamil. Cytotoxcicities of Sea-Nine 211 (EC50 = 99 nM, at 4-arm pluteus) and dichlofluanid (EC50 = 144 nM, at multi-cell) are enhanced by the addition of the P-gp inhibitor, indicating that the two biocides are potential P-gp substrates. Tolylfluanid and CuPT are not transported by P-gp out of the cell, since no obvious changes in the cytotoxicities of the two biocides are observed no matter whether verapamil is added or not. In addition, to understand the mechanisms of ligand binding and its interaction with P-gp, a three-dimensional model of the sea urchin P-gp is generated based on the mouse crystal structure by using homology modeling approach. With this model, a flexible docking is performed and the results indicate that Sea-Nine 211 and dichlofluanid share the same binding site with verapamil, composed of key residues Lys677, Lys753, Thr756, Ala780, Met1033 and Phe1037, whereas tolylfluanid and CuPT display totally different binding modes to P-gp. This further demonstrates that Sea-Nine 211 and dichlofluanid are P-gp substrates, which provides us with new insights into the interactions of P-gp with the antifouling contaminants in aquatic invertebrate embryos.