Background: The microgravity-induced cephalad fluid shift is thought to contribute to neuroophthalmological changes such as optic disc edema, globe flattening, and hyperopic shift. However, the effects of prolonged simulated microgravity on ophthalmic alterations and their potential relationship with functional reorganization in the visual cortex remain unclear. This study aimed to address these knowledge gaps. Methods: A total of 36 participants underwent a 90-day-6 degrees head-down tilt bed rest (HDTBR), a well-established ground-based model for simulating microgravity. Ophthalmic and neuroimaging assessments were performed at three time points: baseline, 1-3 days post-HDTBR, and after a 28-day recovery period. The evaluations included visual function tests such as near visual acuity (NVA), distance visual acuity, best-corrected visual acuity (BCVA), contrast sensitivity, and stereopsis, along with ocular blood flow (OBF) measurements using three-dimensional pseudo-continuous arterial spin labeling (3D-pcASL) and functional connectivity (FC) analysis of the visual cortex via functional magnetic resonance imaging (fMRI). Results: OBF, NVA, and BCVA exhibited a consistent and significant decrease following 90-day HDTBR (all P<0.05). Meanwhile, FC within the primary visual cortex (V1) and the left parietal area F, part M (PFm), as well as between V1 and the visual area 3 (V3), showed a significant increase (voxel level P<0.001, cluster level P<0.025, false discovery rate corrected). Additionally, changes in OBF were positively correlated with alterations in BCVA (r=0.3981, P=0.0162), whereas increased FC between V1 and V3 was associated with a decline in BCVA (r=-0.3394, P=0.0429). Notably, our findings suggest that more than 1 month of recovery may be required to fully counteract these ocular and neural adaptations. Conclusions: OBF may be a key risk factor for decreased visual acuity in stimulated microgravity, potentially driving functional network reorganization of the visual cortex by modifying visual function. These insights contribute to a new insight for ophthalmic health risks associated with human spaceflight.