Cadherins are vital for cell-to-cell interactions during tissue growth, migration, and differentiation processes. Both biophysical and biochemical inputs are generated upon cell-to-cell adhesions, which determine the fate of the mesenchymal stem cells (MSCs). The effect of cadherin interactions on the MSC differentiation still remains elusive. Here we combined the N-Cadherin mimetic peptide (HAV-PA) with the self-assembling E-PA and the resultant N-cadherin mimetic peptide nanofibers promoted chondrogenic differentiation of MSCs in conjunction with chondrogenic factors as a synthetic extracellular matrix system. Self-assembly of the precursor peptide amphiphile molecules HAV-PA and E-PA enable the organization of HAV peptide residues in close proximity to the cell interaction site, forming a supramolecular N-cadherin-like system. These bioactive peptide nanofibers not only promoted viability and enhanced adhesion of MSCs but also augmented the expression of cartilage specific matrix components compared to the nonbioactive control nanofibers. Overall, the N-cadherin mimetic peptide nanofiber system facilitated MSC commitment into the chondrogenic lineage presenting an alternative bioactive platform for stem-cell-based cartilage regeneration.