The peel of Citrus sunki is a rich source of polymethoxyflavones (PMFs). In this study, a PMF-rich fraction (PRF) was obtained from the peels of C. sunki by hot-water extraction and subsequent fractionation with n-hexane. Six PMFs and two demethylated PMFs were isolated by semi-preparative HPLC, and their structures were identified by 1 H NMR and 13 C NMR. To define the synergistic effects of PRF, the PRF and its eight individual components were assessed for antiproliferative activity using HL-60 cells. The PRF showed greater growth inhibitory activity (IC 50 value=25.14μg/ml), compared with its individual components. Flow cytometric analysis confirmed that the PRF significantly increased the percentage of sub-G 1 cells (30.1% at 25μg/ml). Our study provides an efficient method for preparing a PRF for use as a functional food ingredient.