Background: Currently, no systematic evidence synthesis of the mechanism of action of mindfulness-based approaches exists for pain conditions. Aim: To identify and synthesize experimental and clinical studies examining aspects of the mechanism of action of mindfulness for pain relief. Methods: The following databases and search interfaces were searched: Embase (via Embase.com) and Medline (via PubMed). Additional references were identified via bibliographies of included studies. The following were the inclusion criteria applied: (1) original studies published in peer-reviewed journals, (2) in adult populations that (3) examined the mechanism of action of mindfulness meditation on pain outcomes or (4) provided conclusions regarding the potential mechanism of action of mindfulness meditation. The studies were selected by two independent reviewers. Discrepancies were resolved by discussion. Results: A total of 21 studies published in English met the inclusion criteria, of which 5 studies were clinical studies, which included patients with chronic pain, and 16 studies used experimental pain induction. The investigation into brain mechanisms through functional magnetic resonance imaging and diffusion tensor imaging revealed mindfulness meditation's ability to modulate brain activity, particularly in the anterior cingulate cortex, anterior insula, and orbitofrontal cortex, and to enhance structural and functional connectivity in regions associated with pain perception. Regarding the role of opioids, findings across five studies indicated that the analgesic effects of mindfulness are maintained even when opioid receptors are blocked, suggesting a nonopioidergic pathway for pain modulation. Pain perception studies highlighted that mindfulness practices foster pain acceptance and modify pain control beliefs, serving as key mediators in improving pain outcomes. For experienced versus novice mindfulness practitioners, results demonstrated that long-term practice enhances pain threshold and reduces pain unpleasantness through increased activity in salience and attentional control regions. Conclusion: This systematic review highlights mindfulness meditation as a multifaceted approach to pain management, utilizing mechanisms such as cognitive and emotional reappraisal, nonopioidergic pathways, and enhanced attention in control regions. It emphasizes the role of mindfulness in fostering pain acceptance and altering pain control perceptions, showcasing its broad impact on the neurological and experiential dimensions of pain. However, the predominance of studies on healthy subjects and methodological variations across experiments necessitates careful interpretation of the findings. The review calls for further research to explore the mechanisms of mindfulness in chronic pain populations more deeply, distinguishing the specific effects of mindfulness from nonspecific effects and expanding its applicability in clinical settings for chronic pain management.