Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo immunosurgery.