Advanced biofuels from lignocellulosic biomass have been considered as a potential solution for the issues of energy sustainability and environmental protection. Triacylglycerols (TAGs) are potential precursors for the production of lipid-based liquid biofuels. Rhodococcus opacus PD630 can accumulate large amounts of TAGs when grown under physiological conditions of high carbon and low nitrogen. However, R. opacus PD630 does not utilize the sugar L-arabinose present in lignocellulosic hydrolysates. Here, we report the engineering of R. opacus to produce TAGs on L-arabinose. We constructed a plasmid (pASC8057) harboring araB, araD and araA genes derived from a Streptomyces bacterium, and introduced the genes into R. opacus PD630. One of the engineered strains, MITAE-348, was capable of growing on high concentrations (up to 100 g/L) of L-arabinose. MITAE-348 was grown in a defined medium containing 16 g/L L-arabinose or a mixture of 8 g/L L-arabinose and 8 g/L D-glucose. In a stationary phase occurring 3 days post-inoculation, the strain was able to completely utilize the sugar, and yielded 2.0 g/L for L-arabinose and 2.2 g/L for L-arabinose/D-glucose of TAGs, corresponding to 39.7% or 42.0%, respectively, of the cell dry weight.