Diabetic kidney disease (DKD) is a devastating kidney disease and lacks effective therapeutic interventions. The present study was aimed to determine whether reconstituted high-density lipoprotein (rHDL) ameliorated renal injury in eNOS -/- dbdb mice, a mouse model of DKD. Three groups of mice, wild type C57BLKS/J (non-diabetes), eNOS -/- dbdb (diabetes), and eNOS -/- dbdb treated with rHDL (diabetes+rHDL) with both males and females were used. The rHDL nanoparticles were administered to eNOS -/- dbdb mice at Week 16 at 5 μg/g body weight in ~100 μL of saline solution twice per week for 4 weeks via retroorbital injection. We found that rHDL treatment significantly blunted progression of albuminuria and GFR decline observed in DKD mice. Histological examinations showed that the rHDLs significantly alleviated glomerular injury and renal fibrosis, and inhibited podocyte loss. Western blots and immunohistochemical examinations showed that increased protein abundances of fibronectin and collagen IV in the renal cortex of eNOS -/- dbdb mice were significantly reduced by the rHDLs. Taken together, the present study suggests a renoprotective effect of rHDLs on DKD.