Erucic acid (cis-docosa-13-enoic acid, C22:1∆13) and nervonic acid (cis-tetracosa-15-enoic acid, C24:1 ∆15) are important renewable feedstocks in plastic, cosmetic, nylon, and lubricant industries. Furthermore, nervonic acid is also applied to the treatment of some neurological diseases. However, the production of these two very long-chain fatty acids (VLCFA) is very limited as both are not present in the main vegetable oils (e.g., soybean, rapeseed, sunflower, and palm). Ectopic integration and heterologous expression of fatty acid elongases (3-ketoacyl-CoA synthases, KCS) from different plants in Rhodosporidium toruloides resulted in the de novo synthesis of erucic acid and nervonic acid in this oleaginous yeast. Increasing KCS gene copy number or the use of a push/pull strategy based on the expression of elongases with complementary substrate preferences increased significantly the amount of these two fatty acids in the microbial oils. Oil titers in 7-L bioreactors were above 50 g/L, and these two VLCFA represented 20–30% of the total fatty acids. This is the first time that microbial production of these types of oils is reported.