In this study, an oil-producing filamentous fungus, Mortierella elongata MD-3, was obtained from oil-producing plants and their rhizospheres with the aim to develop a new microbial oil resource. The oil content and yield of the strain were 30.15% and 4.67 g/L, respectively. A stable mutant designated MD-3-7 was obtained after treatment of strain MD-3 by atmospheric and room temperature plasma (ARTP), and its oil content and yield were 41.69% and 6.62 g/L, respectively, which increased by 38.27% and 41.76% compared with the original strain. It was found that most of the mycelial pellets of the mutant were villous, and compared to the parental strain, the mycelia became shorter and the density increased, conducive to the accumulation of oil; real-time polymerase chain reaction (real-time PCR) showed that the expression of the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) genes in the mutant strain were up-regulated by 9.25 and 4.37-fold, respectively. This study provides a valuable source for the development and utilization of oil-producing microorganisms.