To investigate the effect of vascularized peroneus longus tendon graft reconstruction on anterior cruciate ligament (ACL) insertion in rabbits. Eighty healthy New Zealand white rabbits were harvested to prepare ACL injury models and randomly divided into two groups ( n=40). The ACL was reconstructed with vascularized peroneus longus tendon graft in group A and peroneus longus tendon graft without blood supply in group B. The survival of animals and the healing of incision were observed after operation; the grafts were taken for gross and histological observations at 4, 8, and 16 weeks; the biomechanical test of the grafts was carried out to record the maximum tensile strength and incidence of ACL insertion rupture at 8 and 16 weeks. All animals survived until the experiment completed. General observation showed that the tunnel was combined with grafts, the vascular infiltration was abundant, and no obvious boundary between the tunnel and grafts existed at 16 weeks in group A; there was still an obvious boundary between the tunnel and graft in group B. Histological observation showed that the collagen fibers between tendon and bone in group A increased gradually, the dense fiber connection was formed, and the "tidal-line" like structure similar to the normal ACL insertion was formed at 16 weeks; but the"tidal-line" like structure was not obvious in group B. Biomechanical test showed that there was no significant difference in the incidence of ACL insertion rupture at 8 and 16 weeks between group A and group B ( P=0.680; P=0.590), but the maximum tensile strength at 8 and 16 weeks were higher in group A than in group B ( t=18.503, P=0.001; t=25.391, P=0.001). The vascularized peroneus longus tendon graft for ACL reconstruction can obviously promote the outcome of the ACL insertion.