The aim of this study was to investigate the effect of the interval between artificial insemination and the onset of estrus on the sex ratio of offspring and fertility in Holstein cows. Standing to be mounted behavior was considered the beginning of estrus, and artificial inseminations were performed 6 h ( Group 1), 9 h (Group 2), 12 h (Group 3), and 15 h (Group 4) after the onset of estrus in cows. Overall 119 (n: 85), 107 (n: 72), 120 (n: 80) and 119 (n: 83) artificial inseminations were performed in Groups 1, 2, 3 and 4, respectively. Pregnancy rates were 67.1%, 70.8%, 72.5% and 79.5% in Groups 1, 2, 3 and 4, respectively (P = 0.33). The highest female ratio of offspring and the pregnancy loss ratios were obtained in Group 2 but this difference was not significant (P = 0.54, P = 0.64). The highest twinning ratio was obtained in Group 4. The number of artificial inseminations per pregnancy was within the economical limits in all groups. In conclusion, the results of this study indicate that the interval between the onset of estrus and artificial insemination time does not alter the sex ratio of offspring in dairy cows with normal fertility parameters.