This study assessed the effect of both embryonic thermal manipulation and dietary threonine level on the response of broilers inoculated with Salmonella Enteritidis, considering bacterial counts in the cecal contents, intestinal morphology, mucin and heat shock protein 70 gene expression, body weight and weight gain. Thermal manipulation was used from 11 days of incubation until hatch, defining three treatments: standard (37.7°C), continuous high temperature (38.7°C) and continuous low temperature (36.7°C). After hatch, chicks were distributed according to a 3x2+1 factorial arrangement (three temperatures and two threonine levels and one sham-inoculated control). At two days of age, all chicks were inoculated with Salmonella Enteritidis, except for the sham-inoculated control group. There was no interaction between the factors on any analyses. High temperature during incubation was able to reduce colonization by Salmonella Enteritidis in the first days, reducing both Salmonella counts and the number of positive birds. It also increased mucin expression and decreased Hsp70 expression compared with other inoculated groups. High temperature during incubation and high threonine level act independently to reduce the negative effects associated to Salmonella Enteritidis infection on intestinal morphology and performance, with results similar to sham-inoculated birds. The findings open new perspectives for practical strategies towards the pre-harvest Salmonella control in the poultry industry.