The study investigates how Sphingosine-1-phosphate receptor 3 (S1PR3) and the Chronic Unpredictable Mild Stress (CUMS) affects depression-like behaviors. The S1P/S1PR3 signaling pathway is known to play a role in mood regulation, but it is not yet fully understood how it is connected to depression. This study looks to further explore this topic. To investigate the effect of CUMS on S1PR3 expression in hippocampus neurons and the synaptic plasticity, we observed animals' behavior with Sucrose Preference Test (SPT), Forced Swim Test (FST) and Open Field Test (OFT). Combining molecular and histological analysis, we investigated the S1PR3 expression, the change in synapse density, and synaptic structure change in the hippocampus. The CUMS caused a significant decrease in the S1PR3 expression, the density of the synaptic spine and synaptic ultrastructure change in mice. On the other hand, over-expression of S1PR3 by adeno-associated virus (AAV) in hippocampal neurons alleviated the depressive-like behaviors and synaptic deficits observed in stress-susceptible animals. Furthermore, the depressive-like phenotype and synaptic impairments were normalized by the expression of RhoA, implicating the RhoA/ROCK1 pathway in S1PR3 actions. Collectively, our findings provide strong evidence that S1PR3 plays a key role in hippocampal synaptic plasticity and depression and that modulation of S1PR3/RhoA/ROCK1 signaling may offer a novel therapeutic strategy for MDD. This study not only underscores the therapeutic potential of S1PR3 but also provides novel insights into the molecular mechanisms underlying depression.