Probiotics reduce stress-related inflammation and abnormal behaviors in humans and rodents via regulation of the microbiota-gut-brain axis. The objective of this study was to determine if probiotic, Bacillus subtilis, has similar functions in broiler chickens under heat stress (HS). Two hundred forty 1-day-old broiler chicks were assigned to 48 pens with 4 treatments: Thermoneutral (TN)-RD (regular diet), TN-PD (the regular diet mixed with 1×106 CFU/g feed probiotic), HS-RD and HS-PD. Probiotic (Sporulin®) was fed from day 1; and HS at 32 ºC for 10 h daily was initiated at day 15. The data showed that final BW, ADG and feed conversion efficiency were improved in PD groups as compared to RD groups regardless of the ambient temperature (P < 0.01). Heterophil to lymphocyte ratio was affected by treatment; and its value was in the order of HS-RD > HS-PD > TN-RD > TN-PD birds (P < 0.01). Compared to TN birds, HS birds spent more time wing spreading, panting, squatting close to the ground, drinking, sleeping, dozing and sitting; but less time eating, standing and walking (P < 0.05 or 0.01). In addition, HS birds had greater levels of hepatic IL-6, IL-10, heat shock protein (HSP)70 and HSP70 mRNA expression (P < 0.01); and greater levels of cecal IgA and IgY (P < 0.01) compared to TN birds. Within TN groups, TN-PD birds had greater concentrations of hepatic IL-10 (P < 0.05) and cecal IgA (P < 0.01) than TN-RD birds. Within HS groups, HS-PD birds spent less time wing spreading, panting, squatting close to the ground, drinking, sleeping, dozing and sitting; but more time eating, foraging, standing and walking than HS-RD birds (P < 0.05 or 0.01). The HS-PD birds also had lower concentrations of hepatic IL-6 and HSP70 (P < 0.01), whereas greater levels of IL-10 (P < 0.05); and lower concentrations of cecal IgA and IgY (P < 0.01). These results indicate that broilers fed the probiotic, bacillus subtilis, are able to cope with HS more effectively by ameliorating heat-induced behavioral and inflammatory reactions through regulation of microbiota-modulated immunity.