Avian pathogenic Escherichia coli (APEC) represents a key pathogen severely threatening the poultry production, as well as food safety and public health. At present, the infection by APEC is hard to be prevented and controlled, because it possesses considerable virulence factors and high insensitivity to antibiotics. Short-chain fatty acids (SCFAs), the primary metabolites of intestinal probiotics, are considered to contribute to maintain intestinal health partially through inhibiting the activity of detrimental bacteria in gut. However, the effects of SCFAs on APEC activity and the underlying mechanisms remain unclear. In this study, we simulated the concentrations of SCFAs in the ileum (i-SCFA) and cecum (c-SCFA) of chickens to investigate their effects on the growth, virulence factor expression, and antibiotic tolerance of APEC. The results indicated that i-SCFA treatment activated the expression of APEC virulence genes, whereas c-SCFA treatment inhibited APEC growth, virulence gene expression, and ampicillin tolerance. Moreover, we observed different response mechanisms of APEC to SCFAs and the equal-pH inorganic acid (hydrochloric acid), which may be attributable to the partial dissociation property and membrane penetration characteristic of SCFAs. Additionally, SCFAs exhibited spatial (intestinal segment-specific) effects on the expression of extracellular and intracellular H