Biomechanical and biological factors can co-dependently influence the establishment of implant-tissue integration; thus, concurrent evaluation of these factors should provide a better understanding of osseointegration. This study aimed to establish and validate an in vivo rat model frequently used in molecular/cellular biology for implant biomechanical studies. We tested the hypotheses that the implant push-in test assesses the degree of osseointegration by the breakpoint load at the implant-tissue interface and that it sensitively differentiates between the effects of different implant surface topographies. The implant push-in test, which produces a consistent load-displacement measurement, was used to test miniature cylindrical titanium implants placed at the distal edge of the adult rat femur. The push-in test values obtained at each post-implantation healing point (weeks 0, 2, 4, and 8) significantly increased in a time-dependent manner. The implant surface after the push-in test was associated with remnant tissues containing host-derived elements, such as calcium, phosphate, and sulfate. In this model, acid-etched implants (average roughness, 0.159 microm) showed significantly greater push-in test values than did turned implants (average roughness, 0.063 microm) throughout the experimental period (p < 0.0001). These results support the validity of the push-in test in rats, which may be used as a rapid and sensitive biomechanical assay system for implant osseointegration research.