INTRODUCTION: The dried seeds of Iris lactea have been used in traditional Chinese medicine. Previous studies have been focused on irisquinones while other chemical components are rarely reported. OBJECTIVE: To establish an efficient high-speed counter-current chromatography (HSCCC) separation method with continuous sample load (CSL) and double-pump balancing (DPB) mode to isolate proanthocyanidins from I. lactea. METHODS: Firstly, an ethyl acetate extract of I. lactea was pre-fractionated by silica column chromatography for the enrichment of proanthocyanidins. Secondly, the enriched proanthocyanidins sample (EPS) was further fractionated by HSCCC with a two-phase solvent system ethyl acetate:n-butanol:water (9:1:10, v/v/v) using DPB mode. The flow rate of the two phases was 2.2 mL/min, the revolution speed was 900 rpm, the separation temperature was 30 °C and the detection wavelength was 280 nm. Finally, the structures of the three isolated proanthocyanidins were elucidated by spectroscopic methods and compared with published data. RESULTS: Under the optimized conditions, 600 mg of the EPS with six continuous injections (100 mg/time) was fractionated, yielding 57 mg of prodelphinidin B3, 198 mg of procyanidin B3, and 162 mg of procyanidin B1, at purities of 97.2%, 98.1% and 97.3%, respectively. CONCLUSIONS: The HSCCC separation method with CSL and DPB proved to be rapid, convenient and economical, constituting an efficient strategy for the isolation of proanthocyanidins.