Urinary plasminogen/plasmin, or plasmin (ogen) uria, has been demonstrated in proteinuric patients and exposure of cultured podocytes to plasminogen results in injury via oxidative stress pathways. A causative role for plasmin (ogen) as a “second hit” in kidney disease progression has yet to have been demonstrated in vivo. Additionally, association between plasmin (ogen) uria and kidney function in glomerular diseases remains unclear. We performed comparative studies in a puromycin aminonucleoside (PAN) nephropathy rat model treated with amiloride, an inhibitor of plasminogen activation, and measured changes in plasmin (ogen) uria. In a glomerular disease biorepository cohort (n = 128), we measured time-of-biopsy albuminuria, proteinuria, and plasmin (ogen) uria for correlations with kidney outcomes. In cultured human podocytes, plasminogen treatment was associated with decreased focal adhesion marker expression with rescue by amiloride. Increased glomerular plasmin (ogen) was found in PAN rats and focal segmental glomerulosclerosis (FSGS) patients. PAN nephropathy was associated with increases in plasmin (ogen) uria and proteinuria. Amiloride was protective against PAN-induced glomerular injury, reducing CD36 scavenger receptor expression and oxidative stress. In patients, we found associations between plasmin (ogen) uria and edema status as well as eGFR. Our study demonstrates a role for plasmin (ogen)-induced podocyte injury in the PAN nephropathy model, with amiloride having podocyte-protective properties. In one of the largest glomerular disease cohorts to study plasminogen, we validated previous findings while suggesting a potentially novel relationship between plasmin (ogen) uria and estimated glomerular filtration rate (eGFR). Together, these findings suggest a role for plasmin (ogen) in mediating glomerular injury and as a viable targetable biomarker for podocyte-sparing treatments.