The current study aimed to assess the effects of dietary alpha-ketoglutarate (AKG) supplementation to laying hens on the fatty acid (FA) profile and cholesterol levels of the egg yolk at the end of production cycle. The experiment was performed on forty-eight Bovans Brown laying hens randomly assigned to either a control group (CONT) or a group supplemented with AKG. The CONT group was fed the basal diet, and the AKG group was fed the basal diet plus 1.0% AKG from the 31st until the 60th week of age, when FA profile, fat and cholesterol content of the egg yolks were determined. No significant changes in the cholesterol and total fat content of the egg yolks were observed. However, there were positive (the decrease in n-6 FA and the increase in MUFA), and negative (decrease in PUFA and n-3 FA, increase in TI and n-6/n-3 ratio) changes in FA profile following AKG supplementation. In conclusion, it was shown that dietary AKG after a 30-week long supplementation influence FA profile in egg yolk and its nutritional value.