Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long‐term resistance to oxidization and low‐temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large‐scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed‐batch fermentation set‐up enable us to reach a specific productivity of 0.032 g·L−1·hr−1 with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.