We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (τ50) of RGD peptides of different flanking residues was found: RGDSPK ≻ RGDSVVYGLR ≈ RGDS ≻ RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily α√-integrin-mediated. In contrast, the τ50 value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both α5β1 and ανβ3 integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides.