Background <p>Multiple myeloma (MM) is a malignant plasma cell tumor. Regulatory T cells (Tregs), key components of the immunosuppressive tumor immune microenvironment (TIME), play a significant role in MM progression. One mechanism of anti-MM activity of anti-CD38 monoclonal antibodies is reducing CD38⁺ Tregs. Cyanidin-3-O-glucoside (C3G), a natural CD38 inhibitor, effectively suppresses enzymatic activity of CD38. Consequently, we investigated whether C3G could serve as a novel therapeutic agent for MM by targeting Tregs.</p> Methods <p>A MM-bearing mouse model was established and treated with C3G to investigate whether C3G can modulate the TIME for therapeutic effects. Tumor growth was monitored, and changes in peripheral blood lymphocyte subsets were assessed to evaluate the immune-modulating effects of C3G. In addition to that, C3G was applied to Naive CD4⁺ T cells derived from the peripheral blood of healthy individuals in vitro to verify its impact on level of Tregs. Network pharmacology and RNA sequencing (RNA-seq) were employed to explore the potential mechanisms by which C3G targets Tregs, and validation was performed using real-time PCR.</p> Results <p>C3G demonstrated therapeutic effects in MM-bearing mice, reducing the proportion of Tregs in peripheral blood while showing no significant effects on T cells, B cells, or NK cells. In vitro studies revealed that C3G’s effects on Tregs were concentration-dependent and capable of reducing Tregs even in the presence of MM cells. Integration of network pharmacology and RNA-seq results suggested that downregulation of granzyme B (<i>GZMB</i>) and upregulation of heme oxygenase 1 (<i>HMOX1</i>) at the transcriptional level are key mechanisms by which C3G influences Tregs.</p> Conclusion <p>C3G exerts therapeutic effects on MM by reducing the proportion of Tregs and thereby restoring anti-tumor immunity. The mechanism may be related to the decrease of <i>GZMB</i> level mediated by the inhibition of CD38.</p>