Previously, we found thymosin β4 (Tβ4) is upregulated in glomerulosclerosis and required for angiotensin II-induced expression of plasminogen activator inhibitor-1 (PAI-1) in glomerular endothelial cells. Tβ4 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 Tβ4 and its prolyl oligopeptidase tetrapeptide degradation product, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), in renal fibrosis. Additionally, we explored interactions of Tβ4 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, Tβ4 plus a prolyl oligopeptidase inhibitor significantly increased fibrosis in wild-type mice. Tβ4 alone also promoted repair and reduced late fibrosis in wild-type mice. Importantly, both profibrotic effects of Tβ4 plus the prolyl oligopeptidase inhibitor, and late reparative effects of Tβ4 alone, were absent in PAI-1 knockout mice. Thus, Tβ4 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 Tβ4 are dependent on PAI-1.