The tunica albuginea (TA) of the penis is an elastic layer that serves a structural role in penile erection. Disorders affecting the TA cause pain, deformity, and erectile dysfunction. There is a substantial clinical need for engineered replacements of TA, but data are scarce on the material properties and biochemical composition of healthy TA. The objective of this study was to assess tissue organization, protein content, and mechanical properties of porcine TA to establish structure-function relationships and design criteria for tissue engineering efforts. TA was isolated from six pigs and subjected to histomorphometry, quantification of collagen content and pyridinoline crosslinks, bottom-up proteomics, and tensile mechanical testing. Collagen was 20 ± 2%/wet weight (WW) and 53 ± 4%/dry weight (DW). Pyridinoline content was 426 ±131 ng/mg WW, 1011 ± 190 ng/mg DW, and 45 ± 8 mmol/mol hydroxyproline. Bottom-up proteomics identified 14 proteins with an abundance of >0.1% of total protein. The most abundant collagen subtype was type I, representing 95.5 ± 1.5% of the total protein in the samples. Collagen types III, XII, and VI were quantified at 1.7 ± 1.0%, 0.8 ± 0.2%, and 0.4 ± 0.2%, respectively. Tensile testing revealed anisotropy: Young's modulus was significantly higher longitudinally than circumferentially (60 ± 18 MPa vs. 8 ± 5 MPa, p < 0.01), as was ultimate tensile strength (16 ± 4 MPa vs. 3 ± 3 MPa, p < 0.01). Taken together, the tissue mechanical and compositional data obtained in this study provide important benchmarks for the development of TA biomaterials. STATEMENT OF SIGNIFICANCE: The tunica albuginea of the penis serves an important structural role in physiologic penile erection. This tissue can become damaged by disease or trauma, leading to pain and deformity. Treatment options are limited. Little is known about the precise biochemical composition and biomechanical properties of healthy tunica albuginea. In this study, we characterize the tissue using proteomic analysis and tensile testing to establish design parameters for future tissue engineering efforts. To our knowledge, this is the first study to quantify tissue anisotropy and to use bottom-up proteomics to characterize the composition of penile tunica albuginea.