The objective of this study was to examine whether an oxytocin challenge test (OCT), evaluated by measuring the changes in uterine blood flow using transrectal colour Doppler ultrasonography, is a suitable non-invasive method to determine uterine contractility in puerperal dairy cows. The changes in uterine blood flow during uterine contractions induced by oxytocin were evaluated on days 2 and 5 postpartum (pp). Twelve clinically healthy Holstein cows were randomly assigned into two groups: (1) oxytocin group (n=7), 50IU oxytocin injected IM and (2) control group (n=5), 5mL saline injected IM. Blood flow volume (BFV) and pulsatility index (PI) in the uterine arteries were determined before and after injection for 120min on days 2 and 5 pp. BFV declined and PI increased rapidly after oxytocin injection on day 2 (P<0.05), whereas oxytocin on day 5 pp did not cause changes in blood flow parameters. The result confirmed that uterine responsiveness to oxytocin decreases with time postpartum in healthy cows. The same OCT was applied in cows with retained fetal membranes (n=6) on day 2 pp, however uterine blood flow showed no change after oxytocin injection. The results showed that an OCT on day 2 pp may be a useful method for investigating the uterine contractile response to oxytocin (reflected as the decrease of uterine blood flow) and the potential pathophysiology of uterine involution in cows.