Previous studies have demonstrated that the modification of the titanium (Ti) surface of an implant with ROD (Arg-Gly-Asp) promotes the activity of osteoblasts. A novel Ti-binding peptide, minTBP-1, and a chimeric peptide, minTBP-1-PRGDN, have been synthesized to assist the fixing of RGD to Ti. In our previous study, minTBP-1-PRGDN demonstrated favorable affinity for Ti surfaces and facilitated the adhesion of MC3T3-E1 cells. The aim of the present study was to evaluate the effect of this chimeric peptide on the proliferation and differentiation of MC3T3-E1 cells. For this purpose, MC3T3-E1 cells were cultured and differentiation was induced on Ti discs precoated with minTBP-1-PRGDN, minTBP-1 or PRGDN. The MC3T3-E1 cells on the minTBP-1-PRGDN-precoated Ti disc were observed to exhibit the highest cell number after 24 h and alkaline phosphatase levels in all groups increased in a time-dependent manner. In addition, marked expression of osteogenic marker genes [osteopontin (OPN) and osteocalcin (OC)] was detected on minTBP-1-PRGDN/Ti at day 14. Mineralized deposits on minTBP-1-PRGDN/Ti presented the maximal average area and the highest number of deposits was observed on PRGDN/Ti. The present study indicates that minTBP-1-PRGDN may enhance and accelerate the activities of MC3T3-E1 cells on Ti, however, its role in vivo must be determined by further studies.