Sphingosine-1-phosphate receptor 2 (S1PR2) is a G-protein-coupled receptor that regulates sphingosine-1-phosphate-triggered cellular response. However, the role of S1PR2 in diabetes-induced glomerular endothelial cell dysfunction remains unclear. This study aims to investigate the effect of S1PR2 blockade on the morphology and function of mitochondria in human renal glomerular endothelial cells (HRGECs). HRGECs were pretreated with a S1PR2 antagonist (JTE-013) or a Rho-associated coiled coil-containing protein kinase 1 (ROCK1) inhibitor (Y27632) for 30 min and then cultured with normal glucose (5.5 mM) or high glucose (30 mM) for 72 h. The protein expression levels of RhoA, ROCK1, and Dynmin-related protein-1(Drp1) were evaluated by immunoblotting; mitochondrial morphology was observed by electron microscopy; intracellular levels of ATP, ROS, and Ca2+ were measured by ATPlite, DCF-DA, and Rhod-2 AM assays, respectively. Additionally, the permeability, apoptosis, and migration of cells were determined to evaluate the effects of S1PR2 and ROCK1 inhibition on high glucose-induced endothelial dysfunction. High glucose induced mitochondrial fission and dysfunction, indicated by increased mitochondrial fragmentation, ROS generation, and calcium overload but decreased ATP production. High glucose also induced endothelial cell dysfunction, indicated by increased permeability and apoptosis but decreased migration. However, inhibition of either S1PR2 or ROCK1 almost completely blocked these high glucose-mediated cellular responses. Furthermore, inhibiting S1PR2 resulted in the deceased expression of RhoA, ROCK1, and Drp1 while inhibiting ROCK1 led to the downregulated expression of Drp1. S1PR2 antagonist modulates the morphology and function of mitochondria in HRGECs via the positive regulation of the RhoA/ROCK1/Drp1 signaling pathway, suggesting that the S1PR2/ROCK1 pathway may play a crucial role in high glucose milieu.