A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme (cellulase and glusulase) at the concentration of 4%, enzymolysis temperature at 30 ℃ and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-screenings,M.isabellina mutant Y-69 was obtained.GC analysis showed that the yield of arachidonic acid by Y-69(2.92g·L-1)was 3.56 times higher than that of the wild-type strain (0.82 g·L-1). Pass generation tests showed that the properties of Y-69 by mutation were readily inherited.