Midichloria mitochondrii is a tick-borne intracellular bacterium of the order Rickettsiales, found with high prevalence in the sheep tick (Ixodes ricinus). Midichloria mitochondrii is capable of vertical transmission in the tick, but recently evidence of potential horizontal transmission to the tick hosts through the blood meal has been reported. We investigated the presence of the bacterium in the blood of roe deer (Capreolus capreolus) collected from an area known to be highly infested with I. ricinus ticks. We collected blood and sera samples for 3 yr in Gardouch (Haute Garonne, France) and subjected them to molecular screening through PCR and to serological investigation using enzyme-linked immunosorbent assay and western blot. Bacterial DNA was detected in the blood of four of seven animals, but only at one or two points in time, whereas all sera were positive for M. mitochondrii antigens at all times. Our results indicated that the presence of the bacterium in the blood is transient, but the antibody response appeared to be long-lasting, possibly due to constant exposure to tick bites, and thus to repeated injection of bacteria. The role of M. mitochondrii in the mammalian host, and its interaction with other tick-borne bacteria, remains unknown.