This study aimed to investigate a protective effect of hydrolyzed wheat gluten (
HWG
) on
Escherichia coli
(
E. coli
)-induced intestinal barrier dysfunctions in broilers. Broilers fed a basal diet unsupplemented or supplemented with HWG (0.5% or 1%) were intraperitoneally injected with either
E. coli
O78 suspension (10
8
CFU/mL) or equal volume of vehicle on d 18 of age. Blood and tissue samples were collected 3rd d post infection. The results showed that
E. coli
-infection increased immune-organ indexes of spleen and thymus, enhanced serum diamine oxidase (
DAO
) level, impaired ileal villus structure and reduced tight junction mRNA levels (
Occludin, Claudin-1, ZO-1, P
< 0.05), while increased mRNA levels of inflammatory cytokines (
TNF-α, IFN-γ, IL-1β, IL-6, IL-8
) and
TLR4
in the ileum of broilers (
P
< 0.05). The effects of
E. coli
O78 challenge on organ indexes of spleen and thymus, serum DAO level, mRNA levels of tight junctions were alleviated by 1% HWG supplementation, the upregulations of
IL-1β
and
TLR4
were prevented by 0.5% HWG supplementation (
P
< 0.05). In addition, increased
IFN-γ
of
E. coli
-infected broilers was abrogated by 0.5% or 1% HWG supplementation (
P
< 0.05). In summary, dietary HWG supplementation ameliorated intestinal barrier dysfunctions triggered by
E. coli
-infection in the ileum of broilers. HWG supplementation might be a nutritional strategy to improve the intestinal mucosal barrier function of broilers.