An improved method for the purification of 1,25-(OH)2D3-26,23-lactone was established by using one step of highperformance liquid chromatography (HPLC). The analytic column was Shim-pack NH2 (straight-phase type). The mobile phase was Methanol: Dichloromethane: Hexane = 4:26:70 and the flow rate was 2ml/min. An ultraviolet detector with 254nm was used. The retention time of 1,25-(OH)2D3, l,25-(OH)2D3-26,23-lactone and 1, 24, 25-(OH)3D3 werel2.10, 17.06 and 22,00 minutes respectively. This method can separate 1,25-(OH)2D3, l,25-(OH)2D3-26,23-lactone and 1,24,25-(OH)3D3 only by one step of HPLC and it is better than the old one which need two steps of HPLC for the separation of these substances.