Background Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal α-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.Methods α-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China.The vector for α-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant α-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of α-galactosidase, we have used it in converting human erythrocytes from group B to group O.Results The purity of recombinant α-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity,metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4℃ in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.Conclusion ECHORBC can be easily obtained from group B red blood cell by α-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.