In this work, we reported a method for the purification of pepsinogens from human gastric mucosa with high pressure gel filtration chromatography (HPLC) and medium pressure anion exchange chromatography (MPLC). First, the two fractions of Pg1 and mixture of PGI and PGII were separated from pepsinogens with HPLC on a Bio-Sil SEC-125 column within 50min. Then the mixture of fractions of PGI and PGII was further separated with MPLC on a Bio-Scale Q2 column within 30min.