The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L⁻¹ depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L⁻¹ of CA with the mass yield coefficient of 0.64 g g⁻¹ and isocitric acid (ICA) amounted to 4.6 g L⁻¹; in the medium with raw glycerol, CA production was 112 g L⁻¹ with the mass yield coefficient of 0.90 g g⁻¹ and ICA amounted to 5.3 g L⁻¹. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.