We investigated the impact of water channels (aquaporin 4; AQP4) on DTI metrics after silencing expression of AQP4 in the juvenile brain. We observed a significant reduction in AQP4 expression after RNA silencing of AQP4 (siAQP4) along with significantly altered DTI metrics in the cortex but not the corpus callosum (CC). No changes in cellular constituents were found. Histological studies have reported decreased AQP4 expression in acquired brain injuries. Thus, our novel findings suggest that reductions in AQP4 expression may underlie the changes in DTI metrics that are often reported in DTI studies of stroke, traumatic brain injury and others.