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Purpose To investigate differences in hippocampal activity between premenstrual syndrome (PMS) patients and healthy controls, to elucidate the neural mechanisms of PMS. Materials and Methods Twenty female patients with PMS (PMS group) and 21 healthy controls (HC group) underwent a single-shot gradient-recalled echo planar imaging (EPI) sequence scan during the luteal phase in 3.0 Tesla MRI. Spontaneous neural activity in hippocampus (HIPP) was measured by fractional amplitude of low-frequency fluctuation (fALFF). Functional connectivity (FC) was used to examine the neural networks of PMS patients by selecting the abnormal HIPP as the seed region. All participants completed a daily record of severity of problems (DRSP) questionnaire to measure the severity of clinical symptoms. Results Results from a two-sample t-test showed increased left HIPP fALFF in the PMS group compared with the HC group (P = 0.042), while there was no between-group difference of fALFF in the right HIPP (P = 0.1011). A secondary analysis using a two-sample t-test with the left HIPP as the seed region, the results revealed that the PMS group exhibited increased FC between the left HIPP and left medial prefrontal cortex (mPFC), left posterior cingulate cortex (PCC), right middle cingulate cortex (MCC), and bilateral precentral cortex (PC), while decreased FC between the left HIPP and right orbitofrontal cortex (OFC). Moreover, the PMS group exhibited higher DRSP scores, which were positively correlated (r = 0.64, P = 0.003) with FC between the left HIPP and mPFC during the luteal phase. Conclusion Altered spontaneous neural activity and connectivity of left HIPP may be involved in PMS. Level of Evidence: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017.