The development of a product that has simultaneous wound healing, anti-inflammatory, and antimicrobial properties is desirable for wound healing medicine. In this study, glycine–histidine–lysine (GHK) peptide as a skin repair accelerator was coupled to L-carnitine with antibacterial and anti-inflammatory properties to investigate its new wound healing properties in a nanoparticle (NP) platform. The conjugate was synthesized by solid-phase synthesis method with Fmoc chemistry, purified by preparative HPLC, and was identified with ESI-Mass technique. The conjugate was then loaded into PLGA NPs, which was prepared using solvent evaporation technique. Zetasizer results showed a mean size and zeta potential of 193.15 nm and −30.2 ± 3.8 mV for the conjugate-loaded PLGA NPs. Scanning electron microscopy (SEM) exhibited spherical-shaped morphology for the NPs with uniform size distribution (PDI = 0.265). Conjugate release from the NPs was about 10% at initial time, which increased to more than about 70% at 200 hr, exhibiting a sustained release trend. Additionally, the treatment of fibroblasts with the nanoconjugate indicated its biocompatibility. Compared to GHK, L-carnitine and free conjugates, the most satisfactory wound healing activity was observed for conjugate-loaded NPs upon wound closure in a rat model of skin injury repair. Furthermore, more epithelialization and neovascularization were observed for L-carnitine–GHK nanoconjugate. Taken together, the L-carnitine–GHK conjugate-loaded PLGA NP will be a promising candidate for wound healing management. [ABSTRACT FROM AUTHOR]