There is an increasing need for the development of functional artificial extracellular matrices (ECMs) for tissue engineering. Recently, we have successfully designed a self-assembling peptide, named E1Y9, to construct functional ECMs. We describe here an enhancement of abilities of E1Y9 materials to promote cell adhesion and differentiation, using functional peptide sequences derived from natural extracellular matrix proteins. We designed functionalized self-assembling peptides, RGDS-conjugated E1Y9 (E1Y9-RGDS) and IKVAV-conjugated E1Y9 (E1Y9-IKVAV). E1Y9-RGDS and E1Y9-IKVAV formed peptide nanofibers in a similar manner to E1Y9, with β-sheet secondary structures. Surfaces coated with peptide nanofibers displayed the higher bioactivities of E1Y9-RGDS for cell adhesion and E1Y9-IKVAV for cell differentiation than those of E1Y9, with the activities being dependent on the concentrations of the functional peptides. These functionalized peptides will be useful for the construction of functional ECMs in cell and tissue engineering. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 731–737, 2013.