Dissolved oxygen (DO) concentration is one of the most important factors affecting microbial cell growth and metabolite formation. In this study, a novel two-stage oxygen supply control strategy was applied to enhance oxygen transfer and promote the ability of arachidonic acid production by Mortierella alpina ME-1. Based on process analysis, a two-stage oxygen supply control strategy was proposed in arachidonic acid production in which n-hexadecane was added as an oxygen vector with a final concentration of 4% at the first of cultivation, the agitation speed was controlled at 180 rpm in the first 96 h, and then switched to 100 rpm. This strategy was experimentally proven to be successful in arachidonic acid fermentation, relatively high arachidonic acid production (15.9 g/L) and dry cell weight (42.5 g/L) was achieved by applying this strategy. The arachidonic acid productivity (2.27 g/L/d) was also successfully improved by 50.3% and 28.9%, respectively, compared with fermentations in which agitation speed was kept constant at 180 rpm and under a two-stage agitation speed control strategy.