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<b><i>Introduction:</i></b> Recently, several mindfulness-based programs showed promising clinical effects in the treatment of psychiatric disorders including substance use disorders. However, very little is known about the effects of mindfulness-based interventions (MBIs) on brain structure in such patients. <b><i>Methods:</i></b> This study aimed to detect changes in gray matter volume (GMV) in opioid-dependent patients receiving MBI during their first month of treatment. Thirty patients were assigned to either 3 weeks of MBI (<i>n</i> = 16) or treatment as usual (TAU, <i>n</i> = 14) and were investigated using structural magnetic resonance imaging before and after treatment. Longitudinal pipeline of the Computational Anatomy Toolbox for SPM (CAT12) was used to detect significant treatment-related changes over time. The identified GMV changes following treatment were related to clinically relevant measures such as impulsivity, distress tolerance, and mindfulness. <b><i>Results:</i></b> After treatment, increased mindfulness scores were found in individuals receiving MBI compared to TAU. In the MBI group, there were also significant differences with respect to distress tolerance and impulsivity. Effects on mindfulness, distress tolerance, and impulsivity were also found in the TAU group. Longitudinal within-group analysis revealed increased left anterior insula GMV in individuals receiving MBI. Anterior insula volume increase was associated with decreased impulsivity levels. In the TAU group, significant GMV changes were found in the right lingual gyrus and right entorhinal cortex. <b><i>Discussion/Conclusion:</i></b> MBI can yield significant clinical effects during early abstinence from opioid dependence. MBI is particularly associated with increased insula GMV, supporting an important role of this region in the context of MBI-induced neural changes.