Abstract: Background and Objective: Probiotics modulate stress-induced changes of physiological homeostasis and behavioral exhibition through regulating the microbiota-gut-brain axis. The aim of this study was to assess if dietary supplementation of probiotic, Bacillus subtilis, reduces aggressive behaviors in laying hens following social challenge. Methodology: Hens (n = 12) of an aggressive stain (Dekalb XL) were housed in single-hen cage prior to the study. At 24 weeks of age, the hens were paired based on their BW to identify the dominance rank within each pair (0 day). The subordinator and dominator of each pair were individually fed a regular layer diet or the diet mixed with 250 ppm probiotic for 2 weeks (days 14). Results: Data showed that the exhibition of aggressive behaviors in the regular diet fed subordinates were not affected by the treatment (p>0.05), while the frequency of threat kick (p = 0.04) was reduced and aggressive pecking (p = 0.053) had a tendency to be lower in the probiotic fed dominates compared to the levels at 0 day. Plasma concentrations of serotonin were also reduced in the probiotic fed dominant hens (p = 0.02). There were no treatment effects on plasma tryptophan levels, body weight gain and egg production (p>0.05, respectively). Conclusion: The data indicate that dietary probiotic supplementation could be a useful management tool for preventing aggressive behaviors in laying hens.