Background <p>Erythropoietin (EPO) is a glycoprotein hormone that exerts pro-angiogenic and anti-inflammatory effects. The present study investigated whether this beneficial profile of action is suitable for improving the in vivo performance of nanofat, an emulsified fat derivative that is clinically used in plastic and reconstructive surgery.</p> Results <p>Repeated intravital fluorescent microscopic analyses showed that EPO-pretreated nanofat significantly accelerates and enhances the vascularization of the implants, as evidenced by an earlier onset of blood perfusion and an increased functional microvessel density when compared to controls. This was associated with a reduced inflammatory response to the implants, as indicated by lower numbers of adherent leukocytes in venules of the host tissue. Histological and immunohistochemical analyses further revealed an improved implant integration with an increased collagen I deposition and a higher density of nanofat-derived CD31⁺/green fluorescent protein (GFP⁺) microvessels, along with a reduced macrophage and neutrophil infiltration. </p> Methods <p>Nanofat was mechanically generated from subcutaneous adipose tissue of GFP⁺ C57BL/6J mice and incubated for 1&#xa0;h in Hank’s Balanced Salt Solution with or without EPO (3 IU/mL). The pretreated nanofat was seeded onto dermal substitutes, which were implanted into dorsal skinfold chambers of GFP⁻ C57BL/6J mice and analyzed over 14 days.</p> Conclusion <p>These findings identify short-term pretreatment with EPO as an effective strategy to boost the vascularization and regenerative capacity of nanofat.</p>