Current rehabilitation for stroke primarily relies on physiotherapy. Research has found that serotonin plays a crucial role in the neuroplastic processes required for brain reorganisation after a stroke. We investigated whether modulation of serotonergic transmission of the dorsal raphe nucleus (DRN), together with rehabilitation training, leads to an improvement in motor recovery.In a photothrombotic stroke mouse model, lesion volume and localization were assessed following 24h after ischemia with 7T-MRI. We chemogenetically activated or inhibited the DRN through adeno-associated virus (AAV) delivery 21 days before the stroke and CNO delivery prior testing sessions. In the critical rehabilitation period of 4 weeks following stroke, automated kinematic analysis of pellet reaching in the staircase, ladder rung, and rotarod test was performed. At the end of the experiment, mice were intracardially perfused, brains were harvested, and sections of the DRN and motor cortex were analysed by immunohistochemistry.Photothrombosis achieved a sufficient stroke volume in the MRI (mean vol 17,3 mm3 +/- 11,4 mm3 SD), that lead to deficits after stroke in all tests. During acute stimulation of the DRN with CNO, mice surprisingly showed a significant decrease in motor performance in the healthy state and after stroke.We observed a drop in motor performance in mice during DRN stimulation, which could be caused by a transient inhibition of motor cortex, that was described in fMRI data in literature. Understanding the potential positive and negative effects of serotonergic modulation on motor recovery after stroke will be important for the understanding of future treatment modalities.