Epimedium koreanum Nakai (E. koreanum) is a traditional herb known for its various health benefits. Especially icariin, a major flavonoid in E. koreanum, has been reported to have pharmacological activities such as antioxidation, antiinflammation, hepatoprotection, immune enhancement and improvement of sexual dysfunction. In this study, we aimed to isolate highly pure icariin from ethanol extract of E. koreanum (EKE) using a DIAION HP-20 column. All fractions separated by HP-20 resin were analyzed using high performance liquid chromatography (HPLC). In order to separate and purify icariin from 100% ethanol extract of E. koreanum, 4 sub-fractions were eluted with 40 % to 100 % ethanol by performing DIAION HP-20 column chromatography. The 60 % ethanol fraction which has the highest content of icariin among four fractions was concentrated and 6 second sub-fractions were eluted with 35 % to 60 % ethanol by performing chromatography. The correlation coefficient for linearity was high with 0.9996 in HPLC method, and the content of icariin was the highest with 28.60 % in 45 % ethanol fraction. As a result, icariin was highly purified by DIAION HP-20 column chromatography. These results suggest that purification using DIAION HP-20 resin can be a suitable method to purify highly pure icariin from EKE. Also, we expect that this method may be used to develop medicines or health-functional foods.