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In the present study, an aqueous extract was prepared using calli from the in vitro-derived leaves of <i>Pyrus pyrifolia</i> cultured in Murashige and Skoog medium containing picloram for a plant growth regulator. The major biological components in the callus extract were identified as uridine (<b>1</b>), adenosine (<b>2</b>), and guanosine (<b>3</b>). In terms of the antioxidant activity, at 300 &#181;g/mL, the extract exhibited free radical scavenging activity of 76.9% &#177; 2.88% in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, comparable to that of 44 &#181;g/mL ascorbic acid (82.5% &#177; 3.63%). In addition, the IC₅₀ values for inhibition of advanced glycation end product formation from collagen and elastin were 602 &#177; 2.72 and 3037 &#177; 102.5 &#181;g/mL, respectively. The extract significantly promoted keratinocyte and fibroblast cell proliferation in a dose-dependent manner. Moreover, fibroblasts treated with 1.36 &#181;g/mL extract exhibited a 1.60-fold increase in procollagen type I C-peptide level compared to controls. The in vitro wound recovery rates of keratinocytes and fibroblasts were also 75% and 38% greater, respectively, than those of serum-free controls at 9 and 36 h after extract treatment (1.36 &#181;g/mL). Additionally, the extract flux across the human epidermis increased by 1598% after its incorporation into elastic nanoliposomes (NLs). Therefore, elastic NLs loaded with <i>Pyrus pyrifolia</i> callus extract have potential use as skin rejuvenators and antiaging ingredients in cosmetic formulations.