Fungi had become the main resource of polyunsaturated fatty acids, especially linoleic acid. The research studied the effects and mechanism of selenium on polyunsaturated fatty acids of Diasporangium jonesianum. The results showed that selenium could significantly increase the yields of linoleic acid. In contrast, the growth and gamma-linolenic acid yield of D. jonesianum was decreased under selenium treatments. Delta 6-Fatty acid desaturase gene of D. jonesianum was investigated in this research. Sequence analysis indicated that this cDNA sequence encoded 235 amino acids. The conserved region of Delta 6-fatty acid desaturase included three conserved histidine-rich domain, hydropathy profile, and was rich in disulfide bonds. This study showed that selenium may in discriminatively substitute S and incorporate selenium-amino acids into the desaturase that the conformation of enzyme active sites was impacted which leaded to the inhibition of the convert of linoleic acid to gamma-linolenic acid and the over accumulation of linoleic acid. Selenium might enhance the fatty acid contents of fungi through influencing the desaturase structure.