Background: Previous studies demonstrated that primary open angle glaucoma (POAG) is associated with abnormal brain structure; however, little is known about the changes in the local synchronization of spontaneous activity. The main objective of this study was to investigate spontaneous brain activity in patients with POAG using regional homogeneity (ReHo) analysis based on resting state functional magnetic resonance imaging (rs-fMRI). Methodology/Principal Findings: Thirty-nine POAG patients and forty-one age-and gender-matched healthy controls were finally included in the study. ReHo values were used to evaluate spontaneous brain activity and whole brain voxel-wise analysis of ReHo was carried out to detect differences by region in spontaneous brain activity between groups. Compared to controls, POAG patients showed increased ReHo in the right dorsal anterior cingulated cortex, the bilateral medial frontal gyrus and the right cerebellar anterior lobe, and decreased ReHo in the bilateral calcarine, bilateral precuneus gryus, bilateral pre/postcentral gyrus, left inferior parietal lobule and left cerebellum posterior lobe. A multiple linear regression analysis was performed to explore the relationships between clinical measures and ReHo by region showed significant group differences in the POAG group. Negative correlations were found between age and the ReHo values of the superior frontal gyrus (r = -0.323, p = 0.045), left calcarine (r = -0.357, p = 0.026) and inferior parietal lobule (r = -0.362, p = 0.024). A negative correlation was found between the ReHo values of the left precuneus and the cumulative mean defect (r = -0.400, p = 0.012). Conclusions: POAG was associated with abnormal brain spontaneous activity in some brain regions and such changed regional activity may be associated with clinical parameters. Spontaneous brain activity may play a role in POAG initiation and progression.