Despite a wealth of data on the effects of spaceflight on tendons and bones, little is known about its effects on the interfacial tissue between these two structures, the enthesis. Mice were sent to space on three separate missions: STS-131, STS-135, and Bion-M1 to determine how spaceflight affects the composition, structure, mechanics, and gene expression of the humerus-supraspinatus and calcaneus-Achilles entheses. At the nanoscale, spaceflight resulted in decreased carbonate levels in the bone, likely due to increased remodeling, as suggested by increased expression of genes related to osteoclastogenesis (CatK, Tnfsf11) and mature osteoblasts (Col1, Osc). Tendons showed a shift in collagen fibril size towards smaller diameters that may have resulted from increased expression of genes related to collagen degradation (Mmp3, Mmp13). These nanoscale changes did not result in micro- and milliscale changes to the structure and mechanics of the enthesis. There were no changes in bone volume, trabecular structure, failure load, or stiffness with spaceflight. This lack of tissue-level change may be anatomy based, as extremities may be less sensitive to spaceflight than central locations such as vertebrae, yet results highlight that the tendon enthesis may be robust against negative effects of spaceflight.