Studies of the microstructure of tendon-bone attachments in mammals have shed light on their role in mediating efficient force transfer between tendon and bone, two tissues that differ vastly in their structure, composition, and material properties. Studies have also elucidated the importance of mechanical stimulation in maintaining tendon-bone attachment structures so that they may function successfully in dissipating mechanical stresses at the tendon-bone interface, to minimize the risk of tissue failure. Gross anatomical studies in fish have focused on identifying pathways of myomere contractile force transmission to the backbone of the fish, via connective tissue septa, to achieve body undulation. Little focus has been placed on how these connective tissues connect to the bone at the microscopic level to achieve force transfer. Moreover, whether or not these attachments are capable of undergoing remodeling in response to novel force regimes—which may influence their function—remains to be elucidated. First, we characterized the microstructure and material properties of the epicentral tendon-vertebra attachment in rainbow trout by conducting histology and tensile-testing. Individual collagen fibers of the epicentral tendon projected directly into the cancellous bone of the vertebra. We observed rostral-to-caudal trends in material properties for attachments found at different positions within the region spanning the dorsal and adipose fin, suggesting that attachments differed in their structure and/or composition at different positions along the backbone. Furthermore, the angle of the epicentral tendon with respect to the bone influenced the material properties and structural integrity of the attachments during tensile-testing, suggesting that attachments may be optimized to transfer force along particular angles. Second, we studied the effect of unloading on the material properties of the epicentral tendon-vertebra attachment by severing a subset of epicentral tendons on one side of rainbow trout. Severing of epicentral tendons had no effect on attachment material properties, suggesting that attachments did not undergo any alterations in structure and/or composition. We propose that the contiguous network of connective tissues in which the epicentral tendons are embedded as well as the fish’s gross musculature may have supported the maintenance of function of the epicentral tendon-vertebra attachments.