A knee joint contracture, a loss in passive range of motion (ROM), can be caused by prolonged immobility. In a rat knee immobilization flexion contracture model, the posterior capsule was shown to contribute to an irreversible limitation in ROM, and collagen pathways were identified as differentially expressed over the development of a contracture. Collagenases purified from Clostridium histolyticum are currently prescribed to treat Dupuytren's and Peyronie's contractures due to their ability to degrade collagen. The potential application of collagenases to target collagen in the posterior capsule was tested in this model.Rats had one hind leg immobilized, developing a knee flexion contracture. After 4 weeks, the immobilization device was removed, and the rats received one 50 µL intra-articular injection of 0.6 mg/mL purified collagenase. Control rats were injected with only the buffer. After 2 weeks of spontaneous remobilization following the injections, ROM was measured with a rat knee arthrometer, and histological sections were immunostained with antibodies against rat collagen types I and III.Compared with buffer-injected control knees, collagenase-treated knees showed increased ROM in extension by 8.0°±3.8° (p-value 0.05). The current study provides experimental evidence for the pharmacological treatment of knee flexion contractures with intra-articular collagenase injection, improving the knee ROM.