The lignin from AFEX-pretreated corn stover and lignin model compounds were evaluated as the sole carbon source for microbial lipid production by Rhodococcus opacus NRRL B-3311. R. opacus NRRL B-3311 could grow on the AFEX-lignin without pretreatment and accumulate lipids up to 32 mg/L in 72 h with consuming 20% of the total lignin. The lipid produced from lignin had a similar fatty acid compositional profile to those of vegetable oil. Based on the metabolism analysis, R. opacus NRRL B-3311 could depolymerize AFEX-lignin and catabolize the aromatic compounds derived from the lignin. Study using aromatic model compounds found that R. opacus NRRL B-3311 preferably utilized 4-hydroxybenzoic acid as the sole carbon source over glucose, while it could not grow on p-coumaric acid or vanillin.