The A adenosine receptor (AR) and D2 dopamine receptor (DR) are two G-protein-coupled receptors that can form dimers and negatively regulate their partners. TAR DNA-binding protein (TDP-43) is a nuclear protein that has been implicated in amyotrophic lateral sclerosis (ALS). Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early step of TDP-43 proteinopathy. Our previous studies indicated that AR is a potential drug target for ALS because treatment with an AR agonist (JMF1907; a T1-11 analog) prevents reactive oxygen species (ROS)-induced TDP-43 mislocalization in a motor neuron cell line (NSC34) and delays motor impairment in a TDP-43 transgenic ALS mouse model. Here， we set out to assess whether activation of DR interferes with the beneficial effects of an AR agonist on motor neurons. We first demonstrated that AR and DR are both located in motor neurons of mouse and human spinal cords and human iPSC-derived motor neurons. Expression of AR and DR in NSC34 cells led to dimer formation without affecting the binding affinity of AR toward T1-11. Importantly， activation of DR reduced T1-11-mediated activation of cAMP/PKA signaling and subsequent inhibition of TDP-43 mislocalization in NSC34 cells. Treatment with quinpirole (a D agonist) blunted the rescuing effect of T1-11 on TDP-43 mislocalization and impaired grip strength in a mouse model of ALS. Our findings suggest that DR activation may limit the beneficial responses of an AR agonist in motor neurons and may have an important role in ALS pathogenesis.