The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R), created by α-1,3-glycosyltransferase (GT), is recognized by natural anti-α-1,3-galactose antibodies in human serum and is responsible for hyperacute rejection (HAR) in pig-to-human/primate xenotransplantation. Although a bioxeno-organ such as heart, kidney, and islet cells from GT knockout (GT KO) pigs has overcome hyperacute rejection, an isoglobotriosylceramide synthase (iGb3s) may produce additional α-1,3-terminated galactose residues on glycosphingolipid. This study was performed to investigate the effect of additional iGb3s KO on the glycoproteomic characterization in the GT^-MCP/-MCP (GT/MCP) transgenic pig cells. The GT^-MCP/-MCP ear fibroblast cells were obtained from GT^-MCP/-MCP pig. The transgenic cells were knocked out with iGb3s and confirmed as GT^-MCP/-MCP+iGb3s^-/- (GT/MCP/iGb3s) cells. WT pig cells were used as a control. To characterize the expression of α-Gal epitope, the both GT/MCP and GT/MCP/iGb3s cells were analyzed by FACS using BS-IB4 lectin antibody. The α-Gal epitope expression in both TG cells was significantly reduced as compared with WT (p<0.05). To analyze a change of glycoprotein levels, the conditioned medium after WT, GT/MCP, and GT/MCP/iGb3s cell culture was collected and performed LC-MS/MS analysis. The data was qualified by GPA (GlycoProteome Analyzer). A total amount of glycopeptide in GT/MCP/iGb3s was increased as compared with that of GT/MCP and WT. However, the glycopeptide forms connected Gal-Gal were reduced in GT/MCP/iGb3s compared to both WT and GT/MCP. On the other hand, the glycopeptides including fucose and sialic acid were increased in GT/MCP/iGb3s compared to both WT and GT/MCP. Our results demonstrated that additional KO of iGb3s combined with GT KO reduced the expressions of α-Gal epitope and glycopeptide forms connecting Gal-Gal. Further studies are needed to product TG piglets and evaluate synergic effects of double gene knock out in order to minimize HAR response after xenotransplantation.