European bison (Bison bonasus) were successfully reintroduced in many free or semi-free areas across Europe during the last decades. Due to the increased numbers, the conflicts between human activities and bison are more frequent. Therefore the knowledge about spatial activity and habitat preference in new regions is the need for management decision making. We studied daily and seasonal habitat use of the semi-free European bison herd in the Czech Republic from 2014 to 2019. The lead cows of the herd were collared with the GPS devices with a 30-minutes GPS fixes interval. The bison herd strongly preferred the managed open areas and supplementary feeding stations during the seasons (Jacob´s index from 0.49 to 0.99). On the contrary, they avoided the forest type and unmanaged open habitats (Jacob´s index from −0.23 to −0.69). The managed meadows and feeding places they used almost exclusively during the night while the forest during the day-light.