In the present study, an aqueous extract was prepared using calli from the in vitro-derived leaves of Pyrus pyrifolia 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 (1), adenosine (2), and guanosine (3). In terms of the antioxidant activity, at 300 µg/mL, the extract exhibited free radical scavenging activity of 76.9% ± 2.88% in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, comparable to that of 44 µg/mL ascorbic acid (82.5% ± 3.63%). In addition, the IC 50 values for inhibition of advanced glycation end product formation from collagen and elastin were 602 ± 2.72 and 3037 ± 102.5 µg/mL, respectively. The extract significantly promoted keratinocyte and fibroblast cell proliferation in a dose-dependent manner. Moreover, fibroblasts treated with 1.36 µg/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 µg/mL). Additionally, the extract flux across the human epidermis increased by 1598% after its incorporation into elastic nanoliposomes (NLs). Therefore, elastic NLs loaded with Pyrus pyrifolia callus extract have potential use as skin rejuvenators and antiaging ingredients in cosmetic formulations.