Although the localization of the proteoglycan/glycosaminoglycan (GAG) complex at the bone-titanium implant interface has been implied, the role of proteoglycans on the establishment of bone-titanium integration is unknown. The hypothesis to be tested was that proteoglycans play an important role in establishing bone-titanium interfacial adhesion. The objective of this study is to investigate the effect of proteoglycan knockdown by GAG enzymatic degradation on the interfacial strength between mineralized tissue and titanium having different surface topographies. Rat bone marrow-derived osteoblastic cells were cultured on either a machined titanium disk or an acid-etched titanium disk. At day 21 of culture, one of the three following GAG degradation enzymes was added into the culture; chondroitinase AC, chondroitinase B, or keratanase. After 3 days of incubation (at day 24 of culture), the laser spallation technique was applied to the samples in order to assess the tissue-titanium interfacial strength. In this technique, a laser-generated stress wave is used to separate the tissue-titanium interface, and the interfacial strength is determined interferometrically by recording the transient free surface velocity of the tissue. Mineralized tissue cultured on the acid-etched titanium showed 20-30% higher tissue interfacial strength than that cultured on the machined titanium (p < 0.0001). For both the machined and acid-etched surface cultures, administration of the enzyme reduced the interfacial strength by 25-30% compared with the untreated control cultures (p < 0.0001). There were no differences in the effect among the three different enzymes tested. A nanoindentation study revealed that the enzyme treatment did not affect the elastic modulus of the mineralized tissue. Scanning electron microscopic and energy dispersive spectroscopic analyses revealed less post-spallation tissue remnant on the titanium substrates when treated with the enzymes. The tissue remnant was greater in amount on the acid-etched surface than on the machined surface. The results suggest that there exists not only mechanical interlocking but also biological interfacial adhesion between the mineralized tissue and titanium, in which the proteoglycan/GAG complex is involved.