Background: manual therapy (MT) has been shown to have positive effects in patients with osteoarthritis (OA)-related pain, and its use in clinical settings is recommended. However, the mechanisms of action for how these positive effects occur are not yet well understood. The aim of the present study was to investigate the influence of MT treatment on facilitatory nociception and endogenous pain modulation in patients with knee OA related pain. Methods: Twenty-eight patients with knee OA were included in this study. Pain intensity using the numerical pain rating scale (NPRS), temporal summation (TS), conditioned pain modulation (CPM), and local (knee) and distant (elbow) hyperalgesia through the pressure pain threshold (PPT), were assessed to evaluate the pain modulatory system. Patients underwent four sessions of MT treatments within 3 weeks and were evaluated at the baseline, after the first session and after the fourth session. Results: the MT treatment reduced knee pain after the first session (p = 0.03) and after the fourth session (p = 0.04). TS decreased significantly after the fourth session of MT (p = 0.02), while a significant increase in the CPM assessment was detected after the fourth session (p = 0.05). No significant changes in the PPT over the knee and elbow were found in the follow-ups. Conclusions: The results from our study suggest that MT might be an effective and safe method for improving pain and for decreasing temporal summation.