We studied birefringence as an indicator of collagen fiber orientation in the diaphysis of the equine third metacarpal bone. We had previously shown that tissue from the lateral cortex of this bone is stronger monotonically, but less fatigue resistant, than tissue from the medial and dorsal regions. To learn whether collagen fiber orientation might play a role in this regional specialization, we tested three hypotheses using the same specimens:​ (1) collagen fiber orientation is regionally dependent;​ (2) remodeling changes collagen fiber orientation;​ (3) longitudinal collagen fibers correlate positively with modulus and monotonic bending strength and negatively with flexural fatigue life. Beams (N=36) cut parallel to the long axes of six pairs of bones had been tested to determine elastic modulus (N=36), and fatigue life (N=24) or monotonic strength (N=12) in four-point bending. Subsequently, histologic cross-sections were prepared, and porosity, active remodeling and past remodeling were quantified. Birefringence was measured as an indicator of transverse collagen orientation using plane-polarized light (PPL), and again using circularly polarized light (CPL). The CPL measurement was less variable than the PPL measurement. Both birefringence measures indicated that collagen was more longitudinally oriented in the lateral cortex than in the other two cortices. Longitudinally disposed collagen correlated with greater modulus and monotonic strength, but did not correlate with fatigue life. Remodeling was associated with more transverse collagen. Neither measure of birefringence was significantly correlated with porosity. It was concluded that, in the equine cannon bone, longitudinal collagen fiber orientation is regionally variable, contributes to increased modulus and strength but not fatigue life, and is reduced by osteonal remodeling.