Understanding how two-dimensional (2D) nanomaterials interact with the biological milieu is fundamental for their development toward biomedical applications. When thin, individualized graphene oxide (GO) sheets were administered intravenously in mice, extensive urinary excretion was observed, indicating rapid transit across the glomerular filtration barrier (GFB). A detailed analysis of kidney function, histopathology, and ultrastructure was performed, along with the in vitro responses of two highly specialized GFB cells (glomerular endothelial cells and podocytes) following exposure to GO. We investigated whether these cells preserved their unique barrier function at doses 100 times greater than the dose expected to reach the GFB in vivo. Both serum and urine analyses revealed that there was no impairment of kidney function up to 1 month after injection of GO at escalating doses. Histological examination suggested no damage to the glomerular and tubular regions of the kidneys. Ultrastructural analysis by transmission electron microscopy showed absence of damage, with no change in the size of podocyte slits, endothelial cell fenestra, or the glomerular basement membrane width. The endothelial and podocyte cell cultures regained their full barrier function after >48 h of GO exposure, and cellular uptake was significant in both cell types after 24 h. This study provided a previously unreported understanding of the interaction between thin GO sheets with different components of the GFB in vitro and in vivo to highlight that the glomerular excretion of significant amounts of GO did not induce any signs of acute nephrotoxicity or glomerular barrier dysfunction.