Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successfully, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol(TAG), and diacylglycerol(DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed detailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result demonstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modi fication of saturated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.