This study was designed to evaluate the role of E. coli α-hemolysin (HlyA) in the pathogenesis of canine pyometra, and on the immune response of canine endometrial epithelial and stromal cells. In Experiment 1, the clinical, hematological, biochemical and uterine histological characteristics of β-hemolytic and non-hemolytic E. coli pyometra bitches were compared. More (p < 0.05) metritis cases were observed in β-hemolytic E. coli pyometra uteri than in non-hemolytic E. coli pyometra uteri. β-hemolytic E. coli pyometra endometria had higher gene transcription of IL-1β and IL-8 and lower gene transcription of IL-6 than non-hemolytic E. coli pyometra endometria (p < 0.01). In Experiment 2, the immune response of endometrial epithelial and stromal cells, to hemolytic (Pyo18) and non-hemolytic E. coli strains (Pyo18 with deleted hlya-Pyo18ΔhlyA- and Pyo14) were compared. Following 4 h of incubation, Pyo18 decreased epithelial cell numbers to 54% (p < 0.001), and induced death of all stromal cells (p < 0.0001), whereas Pyo18ΔhlyA and Pyo14 had no effect on cell numbers. Compared to Pyo18ΔhlyA and Pyo14, respectively, Pyo18 induced a lower transcription level of IL-1β (0.99 vs 152.0 vs 50.9 fold increase, p < 0.001), TNFα (3.2 vs 49.9 vs 12.9 fold increase, p < 0.05) and IL-10 (0.4 vs 3.6 vs 2.6 fold increase, p < 0.001) in stromal cells, after 1 h of incubation. This may be seen as an attempt of hemolytic E. coli to delay the activation of the immune response. In conclusion, endometrial epithelial and stromal cell damage induced by HlyA is a potential relevant step of E. coli virulence in the pathogenesis of pyometra.