The aim of this study was to evaluate the association between estrus’ physical activity at 2 moments (before 30 DIM and at first AI) on ovulation rate and pregnancy per AI (P/AI). A total of 436 lactating Holstein cows were enrolled.Cows were monitored by a pedometer (Afitag, AfiMilk). Ovulation was induced by a timed AI protocol based on estradiol and progesterone. Body condition score (BCS; 1 to 5 scale) was measured at the time of AI (d 0) and the ovaries were scanned on d+7 to check for the presence of a corpus luteum.Calving score and prevalence of endometritis were recorded. Estrus was determined as relative increase(RI) in activity > 100% of the cow’s baseline activity, within the first 30 DIM(30D) and at AI.At estrus, physical activity was categorized as high (<300%RI) and low (<300%RI). Pregnancy was diagnosed at d+30. Data were analyzed using MIXED and GLIMMIX procedures of SAS.Relative increase in activity (mean ± SE) was 274.1 ± 97.3% at 30D estrus and 494.9 ± 159.6% at AI estrus. Low BCS (≤2.75) tended to affect relative increase of activity at 30D (P = 0.09) and at AI (P = 0.12).Milk production was not correlated with increased physical activity (r = 0.06; P = 0.20).Multiparous had lower activity than primiparous at AI (479.8 ± 11.3% vs. 513.1 ± 12.3%; P = 0.04).Cows with endometritis and dystocia had lower activity at 30D estrus compared with those that were healthy or did not experience dystocia (204.3 ± 21.9% vs. 285.7 ± 8.9%; 213.8 ± 26.9% vs. 282.3 ± 13.0%).Cows that had one estrus by 30 DIM had greater P/AI (47.5% vs. 32.8%; P < 0.05) and greater relative increase in activity at AI (533.1 ± 14.8% vs. 477.7 ± 9.9%; P < 0.05) compared with cows with no 30D estrus.Cows with high estrous expression at AI had greater fertility (43.6% vs. 22.8%; P < 0.05) and greater ovulation rates (94.8% vs. 85.7%; P = 0.03).Cows with increased activity at both 30D and AI were more likely to ovulate (98.8% vs. 91.6%; P = 0.01) and had greater P/AI (52.7% vs. 32.9%, P < 0.01) compared with those that did not express estrus at either period. Increase in physical activity at 30D and at AI improved fertility and ovulation rates.