Traditional mycelium aging technology was improved to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1. Filtration step was skipped and additional carbon and nitrogen sources were fed during aging. The levels of the significant factors (time, temperature, ethanol, and KNO(3)) affecting ARA production during improved aging process were also optimized by applying response surface methodology (RSM), and the maximum ARA yield of 19.02 g/l was achieved in a 5 l fermentor at 5.6 days, temperature 13.7 degrees C, ethanol 42.44 g/l, and KNO(3) 2.62 g/l. This yield was 1.55 times higher than that of traditional aging technology. The improved mycelium aging technology is considered to be a useful strategy for enhancing ARA production.