Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re-emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re-examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non-infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.