Ginsenosides Re and Rg1, the main bioactive components of Panax ginseng, have been preparatively isolated and purified using high-speed counter-current chromatography (HSCCC) combined with MCI gel column chromatography (MCI gel CC). Guided by HPLC-MS, the crude extracts from the roots of P. ginseng were pre-separated on an MCI gel column to obtain mixtures of ginsenosides Re and Rg1. Then, a 200 mg volume of enriched sample was separated by HSCCC with a two-phase solvent system composed of ethyl acetate–n-butanol–water (4: 1: 6, v/v/v), yielding 52.6 mg of Rg1 and 37.2 mg of Re with the purities of 99.3% and 98.2%, respectively. The total recovery of Rg1 and Re was 82.4% and 84.4%, respectively, in the two-step separation. This study introduces a fast and efficient method for purification of ginsenosides Re and Rg1 from P. ginseng.