The limitations of current depression treatments highlight the importance of developing new therapeutic strategies. Tanshinone I (Tan I), a naturally occurring lipophilic diterpene compound, has promising activities including inflammation inhibition, cellular autophagy or apoptosis modulation, and anti-oxidative stress. However, the potential antidepressant effects of Tan I and the mechanism behind its action have yet to be established. The antidepressant effect of Tan I was evaluated using animal behavior tests. The chronic unpredictable stress (CUS) mice and C3 overexpressing mice were used to investigate the mechanism of Tan I in microglia-mediated synaptic engulfment, and to explore the effect of Tan I on the improvement of functional magnetic resonance imaging (fMRI)-based network changes in depression-like mice. Here, it is found that Tan I efficiently improved the CUS-induced depressive-like behaviors, attenuated synaptic loss, and inhibited microglial activation. The drug affinity responsive target stability assay and microscale thermophoresis revealed that the specific target of Tan I is complement C3. Furthermore, Tan I decreased the CUS-induced synaptic loss by inhibiting the deposition of C3 deposition onto synapses and subsequent microglia-mediated synaptic engulfment. Importantly, Tan I also improved fMRI-based network changes in CUS mice. Overexpression of C3 in the medial prefrontal cortex (mPFC) of normal mice leads to depressive-like behavior, accompanied by synaptic loss and reduced fMRI-based network changes. In contrast, administration of Tan I inhibits microglia-mediated synaptic phagocytosis and improves fMRI-based network changes, which in turn ameliorate the depressive-like behaviors in C3-overexpressing mice. Collectively, the study demonstrated that Tan I acts as a potent natural C3 modulator that binds directly to C3, blocks the C3-CR3 axis and downstream signal transducer and activator of transcription 3 (STAT3) signaling pathway, inhibits microglia-mediated synaptic engulfment, and improves fMRI-based network changes, which in turn exert antidepressant effects.