Deer antlers offer a unique model to study organ regeneration in mammals. Antler regeneration relies on the pedicle periosteum (PP) cells and is triggered by a decrease in circulating testosterone (T). The molecular mechanism for antler regeneration is however, unclear. Label-free liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify differentially-expressed proteins (DEPs) in the regeneration-potentiated PP (under low T environment) over the non-regeneration-potentiated PP (under high T environment). Out of total 273 DEPs, 189 were significantly up-regulated and 84 were down-regulated from these comparisons: after castration vs before castration, natural T vs before castration, and exogenous T vs before castration. We focused on the analysis only of those DEPs that were present in fully permissive environment to antler regeneration (low T). Nine transduction pathways were identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, including the estrogen signaling pathway. A total of 639 gene ontology terms were found to be significantly enriched in regeneration-potentiated PP (low T) from the DEPs. Reliability of the label free LC-MS/MS was determined by qRT-PCR to estimate the expression level of selected genes. The results suggest that up-regulated heat shock proteins (HSP90AB1, HSP90B1), peptidyl-prolyl cis-trans isomerase 4 (FKBP4), mitogen-activated protein kinase 3 (MAPK3) and calreticulin (CALR) and down-regulated SHC-transforming protein 1 (SHC1), heat shock protein family A member 1A (HSPA1A) and proto-oncogene tyrosine-protein kinase (SRC) may be associated directly or indirectly with antler regeneration. Further studies are required to investigate the roles of these proteins in regeneration using appropriate in vivo models.