This research aimed to evaluate how using betaine levels as a choline substitute affects productive performance, egg quality parameters, fatty acids profile, and antioxidant status in laying hens. One hundred and forty brown chickens, 45 wk old, were divided into 4 groups, each group of 7 replicates with 5 chickens per replicate. The first group of diets with choline has control (A) 100% choline, the second group (B) 75% choline + 25% betaine, the third group (C) 50% choline + 50% betaine, and the fourth group (D) received 100% betaine. No significant effects were observed in final body weight (BW), body weight gain (BWG), egg production (EW), and feed intake (FI) for laying hens. In the diet in which betaine was replaced choline, egg mass (EM) and egg weight (EW) increased compared to the control group (P < 0.05). Also, after 12 wk of feeding, the egg quality parameters were not influenced; however, yolk color was increased significantly compared with the control group. Serum total cholesterol, LDL-lipoprotein, HDL-lipoprotein, triglyceride, glucose, aspartate transaminase (AST), and alanine transaminase (ALT) were not affected by replacing choline with betaine. Furthermore, liver malondialdehyde (MDA) content, yolk vitamin E, and fatty acid levels were not significantly affected by replacing choline with betaine. Moreover, hens fed betaine displayed an increased antibody titer of the Newcastle disease (ND) virus. EW and EM were increased by 3.50% and 5.43% in 100% betaine group (D) when compared to the control group. Isthmus weight was decreased by 48.28 % in 50% choline + 50% betaine group (C) when compared to the control group. ND was increased by 26.24% in 100% betaine group when compared to the control group. In conclusion, betaine supplementation positively affected productive performance, egg quality measurements, and immunity response in Bovans brown laying hens.