Gomisin N, one of the lignan compounds found in Schisandra chinensis (S. chinensis) has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. We proposed a condition of DIAION HP-20 column chromatography, which can isolate highly pure gomisin N from S. chinensis. High performance liquid chromatography (HPLC) was used to analyze the content of gomisin N in isolated fractions. In order to separate and purify gomisin N from 60% ethanol extract of S. chinensis, 3 sub-fractions were eluted with 60% to 100% ethanol by performing DIAION HP-20 column chromatography. 100% ethanol fraction which has the highest content of gomisin N among three fractions was concentrated and 5 second sub-fractions were eluted with 80% to 100% ethanol by performing chromatography. The correlation coefficient for linearity was high with 0.9998 in HPLC method, and the content of gomisin N was the highest with 61.39% in 90% ethanol fraction. As the result, gomisin N was highly purified by DIAION HP-20 column chromatography. The present study may offer a method for separating gomisin N of high purity from S. chinensis ethanol extract. Also, this result shows that it can be used as basic data for producing the highly concentrated products of lignans derived from S. chinensis for a natural product medicine and functional food.