Aims To develop an intrauterine infection model for Trueperella pyogenes in postpartum dairy cows and to assess the effect of this infection on the degree of intrauterine inflammation and concentrations of progesterone in serum. Methods The oestrous cycles of 36 healthy, non-pregnant, postpartum dairy cows were synchronised. They were then treated by intrauterine infusion of 0.5 g cephapirin before being blocked by age and randomly assigned to treatment with intrauterine infusion of saline (n = 18), 10(7) (n = 9) or 10(9) (n = 9) cfu of T. pyogenes, approximately 4 days after the expected time of ovulation (Day 0). Prior to intrauterine infusion on Day 0 and again on Days 3, 7, 10, and 15, cytobrush samples were collected from the uterus of each cow for microbiology and assessment of the percentage of polymorphonuclear neutrophils (PMN%). Blood samples were collected on the same days for measurement of concentrations of progesterone in serum, and uterine lumen diameter was assessed daily using transrectal ultrasonography. Results Trueperella pyogenes was isolated from 5/18 (28%), 7/9 (78%) and 8/9 (89%) cows infused with saline, 10(7) or 10(9) cfu of T. pyogenes, respectively (p < 0.001). Mean PMN% in the control cows did not change over time (p > 0.05), whereas it was higher on Days 7 and 10 than Day 0 in the 10(7) cfu group, and higher on Days 3 and 10 than Day 0 in the 10(9) cfu group (p < 0.05). The percentage of observations with uterine lumen diameters >2 mm was higher in cows infused with 10(7) (29.3 (95% CI = 14.5-44.2)%) or 10(9) cfu (19.2 (95% CI = 7.0-31.5)%) than in control cows (3.1 (95% CI = 0.1-6.0)%) (p < 0.001). Mean concentrations of progesterone in serum were higher in cows infused with 10(7) cfu (2.01 (SE 0.19) ng/mL) than cows infused with 10(9) cfu (1.01 (SE 0.27) ng/mL), with the control group intermediate (1.41 (SE 0.19) ng/mL) (p = 0.03). Conclusions Infusion of 10(7) or 10(9) cfu of T. pyogenes resulted in the establishment of intrauterine infection in 83% of cows. Infection resulted in increased uterine lumen diameter, and an inflammatory response, i.e. elevated PMN%.