High ambient temperature (heat stress,HS) is one of the critical environmental factors causing gut microbiota dysbiosis and increasing gut permeability, consequently inciting neuroinflammation in humans and various animals including chickens. The aim of this study was to examine if a probiotic,Bacillus subtilis, can reduce neuroinflammation in heat-stressed broiler chickens. Two hundred and forty 1-d-old broiler chicks were randomly assigned to 48 pens among 4 treatments in 2 identical, thermal-controlled rooms (n= 12): Thermoneutral (TN)-regular diet (RD), TN-PD(the regular diet mixed with a probiotic at 250 ppm), HS-RD, and HS-PD. The probiotic diet was fed from d 1, and HS at 32°C for 10-h daily was applied from d 15 for a 43-day trial. Results showed that compared to the TN broilers, the HS broilers had higher hippocampal interleukin (IL)-6, toll-like receptor (TLR)4, and heat shock protein (HSP)70 at both mRNA and protein levels regardless of dietary treatment (P< 0.05). In addition, the HS-PD broilers had higher levels of hippocampal IL-8 (P< 0.05) than the TN-PD broilers. Within the HS groups, compared to the HS-RD broilers, the HS-PD broilers had lower levels of IL-6, IL-8, HSP70, and TLR4 (P< 0.05) in the hippocampus. Within the TN groups, the TN-PD broilers had lower IL-8 at both mRNA expressions and protein levels (P< 0.05) but higher TLR4 protein levels (P< 0.05) in the hippocampus as compared to the TN-RD broilers. These results indicate that dietary supplementation of theBacillus subtilis-based probiotic may reduce HS-induced brain inflammatory reactions in broilers via the gut-brain-immune axis. These results indicate the potential use of probiotics as a management strategy for reducing the impact of HS on poultry production.