This study was performed to develop mutant strain using a combination of UV irradiation procedures with protoplast mutagenesis in order to achieve an effective kasugamycin production from Streptomyceskasugaensis. Whenlessthan 1.0g/lof Linoleic acid was used, the cell growth was not inhibited. On the other hand, the cell growth was greatly inhibited when more than 1.6 g/l of linoleic acid was used. Among the various mutant strains, SK-12 was obtained in medium containing 1.6g/l of linoleic acid, showing the highest rate of both cell growth and kasugamycin production. In order to compare kasugamycin production with the SK-12 and the parent strain using soybean oil, cultures were performed in a flask. The production of kasugamycin was increased with the increase time. The maximum kasugamycin concentration was 1.2g/l after 6 days of culture. The product yield from soybean oil was 0.05g/l/g consumed carbon source, which was roughly 5.0 fold higher than the parent strain. These results show that it was effective method for obtaining a mutant resistant to linoleic acid for the effective production of kasugamycin from soybean oil.