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 anti-oxidation, anti-inflammation, hepatoprotection, immune enhancement and improvement of sexual dysfunction. In this study, we aimed to isolate highly pure icariin from the 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. 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 can be used to develop medicines or health-functional foods.