Collagen is an essential component of connective tissue and plays a crucial role in elasticity, stability, and force distribution. While the adaptations of tendon to internal stimuli have been extensively researched, the impact of environmental factors (such as microgravity) on its structure and function at the nanoscopic scale (i.e., fibril level) remains largely unexplored. The purpose of this study was to determine the effects of 32 days (d) aboard the International Space Station (ISS) on murine tendon collagen fibril morphology and nanomechanics (Young’s Modulus, YM). Gastronemius tendon samples from 16wk old female C57BL/6J mice aboard NASA Rodent Research-1 underwent chemical processing and mechanical isolation. Subsequently, collagen fibrils (n equals 17) were studied via atomic force microscopy (AFM, JPK NanoWizard 4a) to compare the morphology (diameter, height) and nanomechanics (average YM) between spaceflight (SF) and ground control (GC) mouse tendon samples. The qp-BioAC-CB1 cantilever (spring constant: 0.3 N/m, tip radius: 10µm) was used under quantitative imaging mode (Qi) for all force and morphological measurements. The data from 17 micrographs (SF n equals 5; GC n equals 12) was processed via the JPK Data Processing Software (Version 6.4.5.), and finally analyzed via Gwyddion (Version 2.62). To our knowledge, this is the first investigation to use AFM QI-mode on tendon collagen fibrils after spaceflight. Mean fibril size and YM were similar between Space Flight (SF) and Ground Control (GC) mouse tendon fibrils after 32 days of space flight; these results suggest that collagen fibrils may require more time to adapt to changes in their environment, and/or more rapid changes may take place at different levels of the collagen hierarchy. This data provides valuable insights into mammalian tissue adaptations during spaceflight, and future research should continue these investigations with longer-duration missions to build a time-course of tendon adaptations to microgravity. This dataset includes results from atomic force microscopy using gastronemius tendon tissue.