It has been suggested that the time of insemination has effect on the calves’ sex ratio because of the differences in timing of capacitation, motility and survival time of the X and Y spermatozoa in the female reproductive tract. We have conducted a field trial to study the effects of different semen deposition sites on the sex ratio and fertility in cattle. Two groups of 450 cows were inseminated via artificial insemination: group A was inseminated into the uterine body and group B was inseminated deep into the uterine horn ipsilateral to the ovary with dominant follicle. After applying several exclusion criteria, a total of 607 pregnant cows were considered for data analysis (group A = 318 and group B = 289 cows). The conception rate was 7% higher (p < 0.05) in the group A, with 23% more of the male calf pregnancies (p < 0.001). At the same time, 18% more of the female calves were calved in the group B (p < 0.005). The difference in male calves between the two groups was 21% and in female calves was 20% (p < 0.001). We conclude that semen deposition site plays a significant role in differences in gender ratio observed after calving. Intracornual semen deposition resulted in a higher ratio of female calves, whereas uterine body deposition site resulted in higher male calves ratio, probably contributing physiologically to the differences in motility, capacitation time, the lifespan of X vs Y spermatozoa and to the pronounced shift of X spermatozoa in the female genital tract.