BACKGROUND: Improving the reproductive performance of dairy cows requires manipulation of estrus using estrus synchronization protocols. OBJECTIVES: The aim of this study was to evaluate the effect of reducing the length of estrus synchronization protocol by using two doses of prostaglandin injection on reproductive performance of Holstein dairy cows. METHODS: At first, all cows received GnRH (day 0), PGF2α on day 7, and GnRH on day 17. Then, cows assigned randomly to one of 3 groups: Group 1) PGF2α injection on days 22 and 23, estradiol benzoate injection on day 24. Group 2) PGF2α injection on days 23 and 24, estradiol benzoate injection on day 25. Group 3) Control group; injection of PGF2α on day 24, injection of estradiol benzoate on day 25. Estrus detection was performed twice a day up to 48 hours (AM/PM) using signs of estrus and fixed time AI protocol was used after 48 hours (if not previously inseminated). RESULTS: Conception rate in group 2 were significantly higher than control group (P≤ 0.05) and tended to be higher than group 1 (P= 0.079). CONCLUSIONS: The results confirmed the hypothesis that a decrease in the period of follicle dominance by reducing the interval between GnRH and PGF2α through using two injections of PGF2α improved fertility of lactating dairy cows.