The effect of probiotic (Lactobacillus sporogenes) feeding on egg production and quality, yolk cholesterol and humoral immune response of White Leghorn (WL) layer breeders was studied. WL layer breeders (24 weeks) were randomly distributed into three groups of 28 each and were housed in individual California type cages. Considering one bird (individual feeding) as a replicate, 28 such replicates were randomly allotted to one of the three dietary treatments: basal (control) or basal with probiotic supplementation at a rate of 100 or 150 mg kg⁻¹ feed. The probiotic used in the study was a commercial preparation containing L. sporogenes with 6000 million spores g⁻¹ of the product. Dietary supplementation of L. sporogenes at 100 mg (6 × 10⁸ spore) kg⁻¹ diet significantly increased egg production and feed efficiency. However, body weight gain, feed intake, egg weight, specific gravity and Haugh unit was not influenced by probiotic feeding. The eggshell breaking strength, shell weight and shell thickness increased significantly by feeding 100 mg probiotic kg⁻¹ diet. The serum concentration of calcium and protein increased, and that of cholesterol decreased by L. sporogenes supplementation. The yolk cholesterol followed the same trend of serum cholesterol. Feeding of L. sporogenes significantly enhanced antibody production. No difference in antibody production could, however, noticed between the levels (100 or 150 mg) of probiotic. It is concluded that feeding of L. sporogenes at 100 mg (6 × 10⁸ spore) kg⁻¹ diet significantly enhanced the egg production, shell quality and humoral immune response of WL layer breeders during 25–40 weeks of age. Copyright © 2007 Society of Chemical Industry