For ACL reconstruction, interference screw can fix the graft in the bone tunnel closer to the articular surface. However, direct interference screw fixation has a possibility to damage the tendon graft at the time of screw insertion, and the bone-tendon contact area is limited within the tunnel. To avoid the damage to the tendon graft at the time of screw insertion and to increase the bone-tendon contact area, a free bone plug was interposed between screw and tendon graft (indirect interference screw fixation). The purpose of this study was to compare ultimate load strength and histological findings between two techniques in a rabbit model. Ultimate pull-out load tests and histological examinations were evaluated at time 0, 3 and 6 weeks. The ultimate failure load of indirect interference screw fixation was significantly higher than that of direct interference screw fixation immediately after surgery (P < 0.05). Histologically, the interface tissues between tendon graft and host bone were more organized and matured in indirect technique. These findings showed that indirect interference screw fixation for tendon graft increased fixation strength at the graft-bone interface, providing quicker graft-bone healing.