Temperament has been associated with reproductive success in beef cattle, with excitable animals often exhibiting reduced fertility. This study evaluated whether acclimating heifers to human handling during an ovulation synchronization protocol improves temperament and pregnancy rates to timed artificial insemination (TAI). A total of 622 Bos taurus yearling beef heifers across five locations and two breeding seasons (eight herd-year observations) were stratified according to reproductive maturity and temperament and were assigned to either acclimation (TRT; n = 307) or control (CTRL; n = 315). Acclimated heifers were moved through handling facilities without restraint prior to each protocol event (days 0, 7, and 10). Temperament was assessed using chute score (CS) and exit velocity (EV), and plasma cortisol was measured in a subset of animals. Acclimated heifers had lower CS on days 7 and 10 (p = 0.011 and p = 0.010, respectively) and greater pregnancy rates to TAI compared with control heifers (54.5% vs. 45.2%; p = 0.018). Exit velocity and cortisol concentrations did not differ between treatments (p ≥ 0.13). These results indicate that acclimation during handling events can improve behavioral responses and pregnancy rates to TAI with modest additional handling time (a mean of 17 s per heifer; no more than 18 min per location/day), providing a practical and scalable strategy for beef producers.