Yarrowia lipolytica can accumulate large amounts of storage lipids and has considerable potential for the production of polyunsaturated fatty acids and other lipids for biofuels. When the nitrogen source is exhausted in the medium, the key intermediate, citrate, is converted to acetyl-CoA by ATP:citrate lyase (ACL) for lipid accumulation. However, in this yeast most of the citrate is also secreted into the culture medium. To increase the endogenous substrate (acetyl-CoA) level for lipid biosynthesis, the acl gene from Mus musculus was over-expressed in Y. lipolytica with mono-copy integration vector pINA1312sp and multi-copy integration vector pINA1292sp. This increased the lipid content from 7.3% to between 11% and 23% (w/w) of the cell dry weight. Cell growth was only slightly affected. Multi-copy integration transformants had higher lipid contents than mono-copy integration transformants; the lipid content of the transformants was consistent with the copy number of acl gene integrated. Over-expression of ACL had no significant effect on fatty acid profile of the yeast. These results suggested that ACL is an important acetyl-CoA producer and plays a vital role in lipid accumulation in oleaginous yeast Y. lipolytica.