ABSTRACT Background Isoproterenol (ISO) is a nonselective beta‐adrenergic receptor agonist known for its vasodilatory effects. This experiment aims to investigate whether intrauterine ISO administration could alter vascular indices and follicular development in postpartum Holstein cows. Objectives The objectives are to evaluate the effects of intrauterine ISO administration on vascular changes and its impact on follicular development compared to placebo groups. Study Design This randomized controlled study was conducted on 36 Holstein cows selected based on their health status, including only those free from reproductive, metabolic and infectious disorders. Methods The cows (n = 36) were divided into two groups as control received distilled water alone (CON, n = 18) and experiment received 4 mg ISO in 40 mL distilled water (ISO, n = 18) and four subgroups as CON‐I (n = 9), CON‐II (n = 9), ISO‐I (n = 9) and ISO‐II (n = 9) according to days of intrauterine administration (I or II represents to 1 or 2 days after ovulation, respectively). Uterine and ovarian artery blood flows were assessed before and after administration by Doppler ultrasonography. Blood samples were collected both before and after administration (on Day 1 or 2) and on Days 3, 6 and 9 post‐ovulation for hormonal analysis. Antral follicle count (AFC) was recorded on the blood sampling days. Data were analysed via mixed model ANOVA. Results Intrauterine ISO administration significantly increased the pulse rate (PR) in the ovaries (89.4 vs. 65.5 bpm, p