The objective of the present study was to evaluate the effects of in ovo infusion of probiotic strains (Bacillus subtilis, Enterococcus faecium, and Pediococcus acidilactici) on jejunal microbial population and mucin gene expression in broiler chicken. In a completely randomized design, 0.5 ml of mediums containing 10 7 cfu of different probiotic strains, was administered into amniotic fluid of the 480 Cobb fetus (day 18 of incubation), with four treatments, five replicates with twenty four eggs each. For mucin gene expression, samples from the jejunum were taken on day 21 of incubation and day 3 post-hatch. Microbial profile was determined for total lactobacillus and E. coli by sampling jejunal contents on days 1 and 3 of age. Expression of the mucin gene in the jejunum was higher (p<0.05) in chicks that received Bacillus subtilis in comparison with the control group. Infusion of the probiotic strains had no effect on jejunal E. coli and lactic acid bacteria populations on day 1 post-hatch (p>0.05). There were no significant differences among treatments for performance parameters at different periods and the whole period. It was concluded that infusion of probiotic bacteria during the late of incubation has no effect on feed intake, gain and feed conversion ratio, but has a positive effect on mucin gene expression in the jejunum. The best probiotic strain for mucin gene expression was Bacillus subtilis and for beneficiary bacteria colonization was Bacillus subtilis and Pediococcus acidilactici.