Objective To investigate the feasibility of establishing an anterior cruciate ligament (ACL) reconstruction model using hamstring tendon autograft in cynomolgus monkeys. Methods Twelve healthy adult male cynomolgus monkeys, weighing 8-13 kg, were randomly divided into two groups (n=6). In the experimental group, the ACL reconstruction model of the right lower limb was prepared by using a single bundle of hamstring tendon, and the ACL of the right lower limb was only cut off in the control group. The survival of animals in the two groups was observed after operation. Before operation and at 3, 6, and 12 months after operation, the knee range of motion, thigh circumference, and calf circumference of the two groups were measured; the anterior tibial translation D-value (ATTD) was measured by Ligs joint ligament digital body examination instrument under the loads of 13-20 N, respectively. At the same time, the experimental group underwent MRI examination to observe the graft morphology and the signal/ noise quotient (SNQ) was caculated. Results All animals survived to the end of the experiment. In the experimental group, the knee range of motion, thigh circumference, and calf circumference decreased first and then gradually increased after operation; the above indexes were significantly lower at 3 and 6 months after operation than before operation (P<0.05), and no significant difference was found between pre-operation and 12 months after operation (P>0.05). In the control group, there was no significant change in knee range of motion after operation, showing no significant difference between pre- and post-operation (P>0.05), but the thigh circumference and calf circumference gradually significantly decreased with time (P<0.05), and the difference was significant when compared with those before operation (P<0.05). At 6 and 12 months after operation, the thigh circumference and calf circumference were significantly larger in the experimental group than in the control group (P<0.05). At 3 and 6 months after operation, the knee range of motion was significantly smaller in the experimental group than in the control group (P<0.05). Under the loading condition of 13-20 N, the ATTD in the experimental group increased first and then decreased after operation; and the ATTD significantly increased at 3, 6 months after operation when compared with the value before operation (P<0.05). But there was no significant difference between the pre-operation and 12 months after operation (P>0.05). There was no significant change in ATTD in the control group at 3, 6, and 12 months after operation (P>0.05), and which were significantly higher than those before operation (P<0.05). At each time point after operation, the ATTD was significantly smaller in the experimental group than in the control group under the same load (P<0.05). The MRI examination of the experimental group showed that the ACL boundary gradually became clear after reconstruction and was covered by the synovial membrane. The SNQ at each time point after operation was significantly higher than that before operation, but gradually decreased with time, and the differences between time points were significant (P<0.05). Conclusion The ACL reconstruction model in cynomolgus monkey with autogenous hamstring tendon transplantation was successfully established.