Aims: This study investigates whether matrix metalloproteinases (MMPs), specifically MMP-2 and MMP-9, interacting with other molecules important in osteoblast differentiation and osteoclastogenesis, could play important roles in the invasion of bone by oral squamous cell carcinoma (OSCC). Methods: Supernatant (conditioned medium, CM) was collected from OSCC cell lines (SCC15 and SCC25), and from cultured osteoblasts (hFOB cell line and a primary culture, OB), and used for indirect co-culture: OSCC cells were treated with CM from osteoblasts and vice versa. Zymogenic activities of MMP-2 and -9, and protein quantities of all molecules studied, were detected by gelatine zymography and Western blotting, respectively. Real-time polymerase chain reaction (PCR) analysed mRNA of these molecules. Targeted molecules were examined by immunohistochemistry in tissue sections of bone-invasive OSCCs. Results: Zymogenic activities of both MMPs were increased in OSCC cells following culture with CM from hFOB: Twist1 protein expression was increased while Runx2 did not alter. The RANKL/OPG ratio, zymogen and protein expression of MMP-9 were increased in hFOB cells cultured with CM from OSCC lines, while zymogen expression of MMP-2 was decreased. Real-time PCR showed generally similar changes in expression of these molecules. All targeted molecules were expressed in invading malignant keratinocytes, and all but OPG were expressed in osteoclasts of clinical samples. Conclusions: Crosstalk between different cell types appears to exist in the invasion of bone by OSCC. Understanding and ultimately interfering with the molecules involved may provide therapeutic approaches to inhibit such bone invasion.