Simple Summary: Ensuring regular reproduction is crucial for successful dairy farming. However, increased milk production demands and efforts to enhance productivity have adversely affected fertility indicators. Fertility disorders are currently significant reasons for culling dairy cows from herds. Accurate estrus detection, based on a complex of physiological signs and behaviors preceding ovulation, is fundamental for achieving desired reproductive outcomes. Successful fertilization hinges on optimal insemination and ovulation timing. The Heatime RuminAct system stands out as a leading technology for estrus detection in cows. It leverages biological principles of cow behavior during estrus, notably, increased locomotion activity. Our study confirms this, showing a substantial (p < 0.001) 33% rise in the locomotion activity of Holstein dairy cows during estrus compared to the reference period 3 days prior, an average increase of 278 u.24 h-1. Post-estrus, locomotion activity dropped significantly on the first day, gradually stabilizing to levels akin to pre-estrus. Through statistical analysis, we explored how parity, lactation stage, milk yield, and individuality affect locomotion activity during estrus and the reference period. Our findings underscored parity's notable impact (F = 13.41, p < 0.001) on altering dairy cows' locomotion patterns during estrus. The objective of this study was the evaluation of the locomotion activity of heifers and Holstein dairy cows during estrus. We have analyzed the locomotion activity using the Heatime RuminAct device on 180 cows (32 heifers and 148 dairy cows) and we evaluated a total of 633 estrus cycles during the reference period of 3 days before estrus, 3 days after estrus, and on the day ofestrus occurrence. The datawere analyzed using the DataFlowTM II program. The locomotion of cows was expressed in the units of locomotion activity in 24 h (u.24 h-1). During the reference period of 3 days before estrus, the cows showed locomotion activity of 558 u.24 h-1, with an increase in locomotion activity on the day of estrus of 836 u.24 h-1, and, during the reference period of 3 days after estrus, the level of locomotion activity decreased to 537 836 u.24 h-1, which is a similar level of locomotion activity to the reference period before estrus. Through the statistical analysis, we evaluated the impact of parity, lactation stage, milk yield, and individuality on changes in locomotion activity during estrus and throughout the reference period, and we found a significant effect of parity (F = 13.41, p < 0.001) on changes in the locomotion activity of dairy cows during estrus. Based on these results, this research offers fresh perspectives on assessing specific factors affecting the locomotion activity of dairy cows during estrus through the practical application of electronic systems for estrus detection on dairy farms.