Background and Purpose: Mitochondrial dysfunction is a driving factor in the development and progression of diabetic kidney disease (DKD). Our laboratory discovered that the 2-adrenoceptor agonist formoterol regulates mitochondrial dynamics in the hyperglycemic renal proximal tubule. Here, we identified signaling mechanisms through which formoterol regulates the mitochondrial fission protein Drp1 and the mitochondrial fusion protein Mfn1. Experimental Approach: Using primary cultures of renal proximal tubule cells (RPTC) exposed to high glucose, we investigated the role of glucose on RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 signaling pathways using pharmacological inhibitors, and the effect of formoterol on these pathways. Key Results: In high glucose, RhoA became hyperactive, leading to ROCK1-induced activation of Drp1. Using pharmacological inhibitors, formoterol signals through G subunits of the 2-adrenoceptor to decrease RhoA/ROCK1-mediated activation of Drp1. Formoterol restored this pathway by preventing the interaction of RhoA with the guanine nucleotide exchange factor p114RhoGEF. Inhibition of RhoA/ROCK1/Drp1 restored maximal mitochondrial respiration. Formoterol also restored Mfn1 through a separate G-dependent mechanism composed of Raf/MEK1/2/ERK1/2/Mfn1. RPTC exposed to high glucose exhibited decreased Mfn1 activation, which was restored with formoterol. Pharmacological inhibition of G, Raf and MEK1/2 also restored Mfn1 activity. Conclusion and Implications: We demonstrate that glucose promotes the interaction between RhoA and p114RhoGEF, leading to increased RhoA/ROCK1/Drp1, and glucose decreases Mfn1 activity through activation of Raf/MEK1/2/ERK1/2. Formoterol restores these pathways and mitochondrial function in response to elevated glucose. Formoterol activates three separate integrative pathways that promote mitochondrial biogenesis, decreased fission and increased fusion in RPTC, supporting its potential as a therapeutic for DKD.