Osteoarthritis (OA) is a degenerative and painful inflammatory joint disease affecting the cartilage, bone, and synovial membranes, without any effective treatment that targets the underlying mechanisms of OA. Our study evaluated the therapeutic effects of a live probiotic strain, Clostridium butyricum GKB7, administered for 6 weeks to rats with knee OA (KOA) induced by anterior cruciate ligament transection (ACLT) of the right knee. All rats underwent weekly weight-bearing behavioral testing and body weight measurements. At 6 weeks, all rats were sacrificed, and the right hind knees were collected for micro-computed tomography imaging and histopathological and immunohistochemical analyses. Compared with rats in the ACLT-only group, ACLT rats administered the probiotic exhibited dramatic improvements in pain-related behavior from postoperative week 2, had significantly less osseous and cartilaginous damage at week 6, and significantly lower levels of the inflammatory markers interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in cartilage and synovium sections. C. butyricum GKB7 appeared to slow or even reverse OA progression and is worth investigating as a novel therapeutic for OA.