Tissue engineering requires the use of polymeric scaffolds that mimic many roles of extracellular matrices (ECM) in the body. Controlling cell–scaffold interactions is one of the most critical parameters for regulating cell phenotype in tissue engineering, and a peptide with the sequence of RGD has been widely exploited for this purpose. We hypothesized that the spacer arm length of adhesion ligands coupled to synthetic ECMs could be vital for regulation of cell–scaffold interactions. We prepared alginate gels modified with RGD peptides containing varying spacer arm lengths and cultured primary human fibroblasts either on the gels (2-D) or within the gels (3-D). The spacer arm length of the RGD peptides significantly influenced the adhesion and proliferation of fibroblasts in both the 2-D and 3-D studies. We found that a minimum number of four glycine units in the spacer arm was essential for enhanced adhesion and growth of the cells in vitro. An optimal spacer arm length of the RGD peptides was also necessary for minimizing cellular stress responses as determined by analyzing expression of heat shock proteins and Bcl-2 in cultured cells. This approach to controlling cell phenotype using adhesion peptides with various spacer arm lengths could be useful for designing novel scaffolds in tissue engineering applications.