Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family and a ligand for the tropomyosin-receptor kinase B (TrkB), mediates neuronal survival, differentiation, and synaptic plasticity. However, BDNF is not used to treat neurodegenerative diseases because of its poor pharmacokinetic profile, side effects, and absence of survival properties in clinical trials. Consequently, alternative approaches such as TrkB receptor agonist application are gaining importance. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has been described as a robust TrkB receptor agonist in hippocampal neurons. Nevertheless, the influence of 7,8-DHF on motoneurons, one of the main targets of BDNF in vivo, is so far unknown. Therefore, we investigated the impact of 7,8-DHF treatment on primary cultured mouse motoneurons. Indeed, we found an activation of the TrkB receptor. Moreover, 7,8-DHF application promotes survival and neurite growth of cultured motoneurons and these effects appear dose-dependent. To investigate the PI3K/AKT and MAPK pathway activation in 7,8-DHF treated motoneurons, we developed a high-density culture system of primary mouse motoneurons. Analysis of both pathways demonstrated a PI3K/AKT but not MAPK pathway activation in cultured motoneurons. This is in contrast to previously published reports about BDNF-mediated activation of TrkB. The lack of MAPK pathway activation is also in contrast to what has been found for hippocampal neurons that indeed show MAPK activation after 7,8-DHF treatment. The ability of 7,8-DHF to imitate BDNF function in motoneurons by using Trk receptor signaling would provide a new approach for the treatment of motoneuron diseases, but needs a more detailed analysis of the activation profile of 7,8-DHF.