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The effect of the inseminating dose of fresh sex-sorted semen on pregnancy per artificial insemination (P/AI) in lactating dairy cows was evaluated. Semen from 3 Angus and 3 Simmental bulls was used. Sex-sorted Y-chromosome–bearing sperm was split into 4 aliquots and extended to achieve (1) 2 × 106 spermatozoa of fresh sex-sorted semen per straw (Fresh-2M), (2) 4 × 106 spermatozoa of fresh sex-sorted semen per straw (Fresh-4M), (3) 6 × 106 spermatozoa of fresh sex-sorted semen per straw (Fresh-6M), and (4) 4 × 106 spermatozoa of frozen sex-sorted semen per straw (Frozen-4M). At 34 d post AI, P/AI differed among groups (35.4% vs. 40.0% vs. 39.9% vs. 34.7% for Fresh-2M vs. Fresh-4M vs. Fresh-6M vs. Frozen-4M, respectively). Increasing the dose of fresh sex-sorted semen from 2 × 106 to 4 × 106 spermatozoa increased P/AI. Cows inseminated with Fresh-4M and Fresh-6M had more P/AI compared with cows inseminated with Fresh-2M and more P/AI compared with cows inseminated with Frozen-4M. An asymptotic relationship between number of spermatozoa inseminated and P/AI was identified. The predicted probability of pregnancy increased rapidly with increasing semen dose up to ∼2.3 × 106 spermatozoa, after which the response approached an asymptote, with minimal additional improvement at higher doses. In conclusion, insemination with 4 × 106 spermatozoa of fresh sex-sorted semen optimized fertility in lactating dairy cows, with no additional benefit from increasing the dose to 6 × 106 spermatozoa of sex-sorted semen. Based on the dose-response model, 99% of maximal fertility was achieved at an estimated dose of 3.5 × 106 spermatozoa of fresh sex-sorted semen.