Cannabis use typically begins during adolescence and early adulthood, a period when cannabinoid receptors are still abundant in white matter pathways across the brain. However, few studies to date have explored the impact of regular cannabis use on white matter structure, with no previous studies examining its impact on axonal connectivity. The aim of this study was to examine axonal fibre pathways across the brain for evidence of microstructural alterations associated with long-term cannabis use and to test whether age of regular cannabis use is associated with severity of any microstructural change. To this end, diffusion-weighted magnetic resonance imaging and brain connectivity mapping techniques were performed in 59 cannabis users with longstanding histories of heavy use and 33 matched controls. Axonal connectivity was found to be impaired in the right fimbria of the hippocampus (fornix), splenium of the corpus callosum and commissural fibres. Radial and axial diffusivity in these pathways were associated with the age at which regular cannabis use commenced. Our findings indicate long-term cannabis use is hazardous to the white matter of the developing brain. Delaying the age at which regular use begins may minimize the severity of microstructural impairment.