Aims To evaluate the specific role of the endocannabinoid/cannabinoid type‐1 (CB 1 R) system in modulating mitochondrial dynamics in the metabolically active renal proximal tubular cells (RPTCs). Materials and methods We utilized mitochondrially‐targeted GFP in live cells (wild‐type and null for the CB 1 R) and electron microscopy in kidney sections of RPTC‐CB 1 R ‐/‐ mice and their littermate controls. In both in vitro and in vivo conditions, we assessed the ability of CB 1 R agonism or fatty acid flux to modulate mitochondrial architecture and function. Results Direct stimulation of CB 1 R resulted in mitochondrial fragmentation in RPTCs. This process was mediated, at least in part, by modulating the phosphorylation levels of the canonical fission protein dynamin‐related protein 1 on both S637 and S616 residues. CB 1 R‐induced mitochondrial fission was associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we documented that exposure of RPTCs to a fatty acid flux induced CB 1 R‐dependent mitochondrial fission, lipotoxicity and cellular dysfunction. Conclusions CB 1 R plays a key role in inducing mitochondrial fragmentation in RPTCs, leading to a decline in the organelle's function and contributing to the renal tubular injury associated with lipotoxicity and other metabolic diseases.