Collagen, the major extracellular matrix component, is a popular biomaterial due to its excellent biocompatibility. Collagen is widely used in sutures, corneal bandages, drug delivery gels, and as wound dressings, as well as in a newly emerging interdisciplinary biomedical field, tissue engineering. A better understanding of the interaction between cells and collagen substrata combining other ECM components should allow for a more rational scaffold design for tissue engineering. In this study, we explored the effects of heparin on the behavior of osteoblast-like cell lines (MG-63) on collagen-based substrates. Collagen mixture (0.1 mg/mL) with heparin at different concentrations (0.02 mg/mL, 0.1 mg/mL, and 0.5 mg/mL) was coated onto tissue culture polystyrene plates. We found that low concentration of heparin (0.02 mg/mL) enhanced cell adhesion and spreading on collagen-coated plates, but did not influence cell growth and alkaline phosphatase activity. On the other hand, high doses of heparin (0.1 and 0.5 mg/mL) decreased cell adhesion and spreading, as well as cell proliferation. Our results suggest that a small amount of heparin might support osteoblast attachment and spreading although high heparin concentration is not suitable for cell culture.