The objective of this study was to determine the effect of timing of artificial insemination on pregnancy rates, calving rates, abortion rates, twinning rates, and calf gender ratio after synchronization of ovulation with Ovsynch protocol in Holstein dairy cows. The ovulation of 219 lactating Holstein dairy cows was synchronized using the Ovsynch protocol. Therefore, cows received an injection of GnRH followed by an injection of prostaglandin F(2α) 7 days later and a second treatment with GnRH 2 days later. Cows were artificially inseminated at 0, 12, or 24 h after the second injection of GnRH. Reproductive performance did not differ between cows inseminated at 0 h (n = 82), 12 h (n = 66), or 24 h (n = 71) after the last injection of GnRH (pregnancy rate: 0 h 48 %, 12 h 47 %, 24 h 52 %; abortion rate: 0 h 5 %, 12 h 0 %, 24 h 11 %; calving rate: 0 h 43 %, 12 h 47 %, 24 h 41 %; twinning rate: 0 h 2 %, 12 h 0 %, 24 h 0 %; calf gender ratio (F/M): 0 h 61:39 %, 12 h 48:52 %, 24 h 39:61 %; P > 0.05). Pregnancy rates for cows inseminated in postpartum times of 50-75, 76-100, and >100 days within the first and ≥ 3 parities were statistically similar (P > 0.05), but pregnancy rates for cows inseminated at different postpartum times of 50-75, 76-100, and >100 days within the second parity were different (P < 0.01). In general, pregnancy rates of cows inseminated at different postpartum times (P < 0.01) and parities (P < 0.001) differed. The findings of the current study showed that rates of pregnancy, abortion, calving, and twinning of Holstein dairy cows subjected to artificial insemination at different times after synchronization were similar. These results also indicate that the timing of artificial insemination after synchronization did not influence calf gender ratio. Furthermore, pregnancy rates of Holstein dairy cows inseminated after synchronization were significantly influenced by postpartum time and parity number.