Previously, we found thymosin beta 4 (Tb4) is upregulated in glomerulosclerosis and required for angiotensin II-induced expression of plasminogen activator inhibitor-1 (PAI-1) in glomerular endothelial cells. T beta 4 has beneficial effects in dermal and corneal wound healing and heart disease, yet its effects in kidney disease are unknown. Here we studied renal fibrosis in wild-type and PAI-1 knockout mice following unilateral ureteral obstruction to explore the impact of T beta 4 and its prolyl oligopeptidase tetrapeptide degradation product, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), in renal fibrosis. Additionally, we explored interactions of T beta 4 with PAI-1. Treatment with Ac-SDKP significantly decreased fibrosis in both wild-type and PAI-1 knockout mice, as observed by decreased collagen and fibronectin deposition, fewer myofibroblasts and macrophages, and suppressed profibrotic factors. In contrast, T beta 4 plus a prolyl oligopeptidase inhibitor significantly increased fibrosis in wild-type mice. T beta 4 alone also promoted repair and reduced late fibrosis in wild-type mice. Importantly, both profibrotic effects of T beta 4 plus the prolyl oligopeptidase inhibitor, and late reparative effects of T beta 4 alone, were absent in PAI-1 knockout mice. Thus, T beta 4 combined with prolyl oligopeptidase inhibition is consistently profibrotic, but by itself has antifibrotic effects in late-stage fibrosis, while Ac-SDKP has consistent antifibrotic effects in both early and late stages of kidney injury. These effects of T beta 4 are dependent on PAI-1.