The solid fermentation conditions, including fermentation medium, fermentation time, initial pH, as well as additional carbon and nitrogen source for borrelidin produced by Streptomycete rochei ATCC10739 were investigated. Under the optimal fermentation conditions (ISP-2 medium supplemented with 1% glycerol; initial pH 6.0; culture temperature 30 °C; fermentation time, 7 days), the highest borrelidin production level was achieved as 1.336 mg/L media. Borrelidin was separated and purified by organic extraction, followed by silica gel chromatography and semipreparative high performance liquid chromatography (HPLC).