Tendons play a critical role in the transmission of forces between muscles and bones, and chronic tendon injuries and diseases are among the leading causes of musculoskeletal disability. Little is known about sex‐based differences in tendon structure and function. Our objective was to evaluate the mechanical properties, biochemical composition, transcriptome, and cellular activity of plantarflexor tendons from 4 month old male and female C57BL/6 mice using in vitro biomechanics, mass spectrometry‐based proteomics, genome‐wide expression profiling, and cell culture techniques. While the Achilles tendons of male mice were approximately 6% larger than female mice (p < 0.05), the cell density of female mice was around 19% greater than males (p < 0.05). No significant differences in mechanical properties (p > 0.05) of plantaris tendons were observed. Mass spectrometry proteomics analysis revealed no significant difference between sexes in the abundance of major extracellular matrix (ECM) proteins such as collagen types I (p = 0.30) and III (p = 0.68), but female mice had approximately twofold elevations (p < 0.05) in less abundant ECM proteins such as fibronectin, periostin, and tenascin C. The transcriptome of male and female tendons differed by only 1%. In vitro, neither the sex of the serum that fibroblasts were cultured in, nor the sex of the ECM in which they were embedded, had profound effects on the expression of collagen and cell proliferation genes. Our results indicate that while male mice expectedly had larger tendons, male and female tendons have very similar mechanical properties and biochemical composition, with small increases in some ECM proteins and proteoglycans evident in female tendons. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2117–2126, 2017.