Abstract The objective of the present experiment was to investigate the effect of monensin (MN) on the time course of recovery from diet-induced milk fat depression. Milk fat depression was induced in all cows (n = 16) during the first phase of each period by feeding a low-fiber, high-unsaturated fat diet [25.3% neutral detergent fiber (NDF), 6.9% fatty acids (FA), and 3.24% C18:2] with MN (450mg/cow per day) for 10 to 14d. A recovery phase of 18d followed, where cows were switched to a higher-fiber and lower unsaturated fat diet (31.2% NDF, 4.3% FA, and 1.7% C18:2). According to a crossover design, treatments during recovery were (1) control (no MN supplementation) or (2) continued MN supplementation. Milk yield, milk composition, and milk FA profile were measured every 3d during recovery. No effect was observed of MN on dry matter intake or yield of milk, milk protein, and lactose. Milk fat concentration and yield increased progressively during recovery in both treatments. Monensin decreased milk fat yield from d 6 to 15, but it was the same as the control on d 18. A treatment by time interaction on milk fat concentration was detected, which was decreased by MN only on d 3 and 6. The yield of milk de novo synthesized FA increased progressively in both treatments and was not affected by treatment. Similarly, yield of 16-C FA increased progressively, but was decreased by MN on d 6 and 9. Preformed FA yield was lower in the MN group from d 6 to 15, but was not different from the control on d 18. Importantly, milk FA concentration of trans-10 C18:1 and trans-10,cis-12 conjugated linoleic acid rapidly decreased in both groups; however, MN slightly increased trans-10 C18:1 concentration above baseline on d 15 and 18. In conclusion, MN supplementation had minimal effect on recovery of normal rumen biohydrogenation and de novo FA synthesis during recovery from milk fat depression by correction of dietary starch, NDF, and polyunsaturated FA concentration, but moderately decreased recovery of preformed FA in milk.