Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99–100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.