Abstract: AIM　To elucidate the microheterogeneity of three N-linked oligosaccharide sites of the Chinese-made recombinant human erythropoietin (rHuEPO). METHODS　Glu-C digestion, RP-HPLC separation, online HPLC/electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS　The sialic acid was analyzed directly. Almost every oligosaccharide was acetylated, the acetylation of tetraantennary+2LacNAc+4SA and tetraantennary+2LacNAc+4SA were reported. CONCLUSION　The acetylation of multi-antennary oligosaccharide will improve the activity of rHuEPO in vivo. The biantennary oligosaccharide was found mainly existing at N-24. For the first time, the carbohydrate structures of each N-linked glycosylated site of Chinese-made rHuEPO were reported.