Betaine, an important methyl donor, is known to execute epigenetic regulation of gene expression via nutritional reprogramming. Herein, we explore whether feeding a betaine-supplemented diet to laying hens would affect corticosteroid biosynthesis in the adrenal gland and corticosterone deposition in eggs, in correlation with the expression of methyl transfer enzymes and the promoter DNA methylation status of affected genes. Rugao yellow-feathered laying hens at 38 weeks of age were assigned to Control and Betaine groups, fed basal and betaine-supplemented diets, respectively, for four weeks. Betaine supplementation significantly increased (P < 0.05) the average laying rate, while the body weight and egg quality remained unchanged. Plasma concentrations of cholesterol and low-density lipoprotein-cholesterol were also higher (P < 0.05) in the Betaine group. Moreover, eggs in the Betaine group contained higher corticosterone in the yolk, which was associated with up-regulation of steroidogenesis genes in adrenal glands. Steroidogenic acute regulatory protein (StAR), the rate-limiting protein responsible for transporting cholesterol to the inner mitochondrial membrane, was significantly activated (P < 0.05), together with its transcription factors steroidogenic factor-1 (SF-1) and glucocorticoid receptor. Also, betaine supplementation significantly up-regulated (P < 0.05) the adrenal mRNA expression of adenosyl homocysteinase-like 1 and DNA methyltransferases1 and 3a. Bisulfite sequencing analysis revealed significant hypomethylation in several CpG sites within the promoter region of SF-1 gene in the adrenal gland. These results indicate that dietary supplementation of betaine in hens activates adrenal expression of StAR, possibly through epigenetic regulation of SF-1 gene.