Engineering synthetic hydrogels on a molecular basis to introduce natural features that are important in instructing cell behavior is becoming increasingly crucial in biomaterial-based approaches for regenerative medicine and in cell biology to study cell–matrix interactions in three-dimensions (3D). Here, we used collagen gels and exploited the design flexibility of the biological, biochemical and physical characteristics offered by a PEG-based hydrogel system to systematically study the effect of specific extracellular microenvironments on the behavior of primary human fibroblasts in 3D. We firstly found that the proliferation profiles of fibroblasts from different patients cultured within collagen gels (3D) differed significantly from their behavior observed on tissue culture plastic (2D). Furthermore, using the biomimetic PEG-based matrix we showed that cell proliferation in 3D could be selectively manipulated via alteration of the gel characteristics. In particular, this study revealed that, in spite of matrix sensitivity to proteases (e.g. MMP) and the presence of cell-integrin binding sites, at high stiffness (elastic modulus, G′ >1200 Pa) the matrix acts as a barrier for cells cultured in 3D. Finally, a comparison between the biomimetic PEG-based and collagen gels indicated that differences in their viscoelastic behaviours, determined by the nature of network structures and cross-links, may influence the mechanism(s) cells employ to remodel their 3D extracellular microenvironment. In conclusion, these studies highlight that for proliferation in 3D, compared to 2D, cells require strategies to overcome the physical impediment posed by the matrix. We also demonstrate that by exploiting the design flexibility of the characteristics offered by these biomimetic hydrogels, it is possible to separately investigate complex aspects characterizing the cell–matrix interactions in 3D; this has the potential to have great impact in regenerative medicine, as well as in cell biology and cancer research.