Microalgae are one of the most promising feedstocks for biodiesel production due to their high lipid content and easy farming. However, the extraction of lipids from microalgae is energy intensive and costly and involves the use of toxic organic solvents. Compared with organic solvent extraction, supercritical CO2 (SCCO2) has demonstrated advantages through lower toxicity and no solvent-liquid separation. Due to the nonpolar nature of SCCO2, polar organic solvents such as methanol may need to be added as a modifier in order to increase the extraction ability of SCCO2. In this paper, pilot scale lipid extraction using SCCO2 was studied on two microalgae species: Spirulina sp. and Schizochytrium sp. For each species, SCCO2 extraction was conducted on 200 g of biomass for 6 h. Methanol was added as a cosolvent in the extraction process based on a volume ratio of 4%. The results showed that adding methanol in SCCO2 increased the lipid extraction yield significantly for both species. Under an operating pressure of 4000 psi, the lipid extraction yields for Spirulina sp. and Schizochytrium sp. were increased by 80% and 72%, respectively. It was also found that a stepwise addition of methanol was more effective than a one-time addition. In comparison with Soxhlet extraction using methylene chloride/methanol (2:1, v/v), the methanol-SCCO2 extraction demonstrated its high effectiveness for lipid extraction. In addition, the methanol-SCCO2 system showed a high lipid extraction yield after increasing biomass loading fivefold, indicating good potential for scaling up this method. Finally, a kinetic study of the SCCO2 extraction process was conducted, and the results showed that methanol concentration in SCCO2 has the strongest influence on the lipid extraction yield.