Mesenchymal stem cells (MSCs) are an essential tool in cell-based therapies. One of the most crucial factors for efficacy in regenerative medicine is the source of MSCs. Tissue origin has long been suggested as a potential determinant of MSC properties. Human dermal fibroblasts share similar characteristics to MSCs, and the question of whether fibroblasts are functionally equivalent to MSCs is still debated. The present work used proteomic and phenotypic analyses to compare human dermal fibroblasts, dental pulp stem cells (DPSCs), and adipose-derived mesenchymal stem cells (AD-MSCs). We observed similarities and differences in morphology, cell surface markers, differentiation, and proteomic profile. Proteome was profiled by nano liquid chromatography and comprehensively quantified by ion mobility-enhanced mass spectrometry. Fibroblasts and MSCs shared similar surface markers, growth kinetics, and differentiation capacity. Proteomic analysis reproducibly identified and quantified 3051 proteins, 109 of them were differentially abundant according to strict statistic criteria. We showed that CD13, CD9, and CD105 could potentially distinguish MSCs of different origin and fibrobrasts. GO term and GSEA enrichment analysis of differentially accumulated proteins identified signaling pathways characteristic to individual cell types. Particularly, we highlighted signaling pathways involved in cell migration, adhesion, and Wnt signaling as downregulated in fibroblasts in comparison to DPSCs. Angiogenesis and vascularization were specifically associated with AD-MSCs.