The mechanical properties of cortical bone vary not only from bone to bone;​ they demonstrate a spatial viability even within the same bone due to its changing microstructure. They also depend considerably on different loading modes and orientations. To understand the variability and anisotropic mechanical behavior of a cortical bone tissue, specimens cut from four anatomical quadrants of bovine femurs were investigated both in tension and compression tests. The obtained experimental results revealed a highly anisotropic mechanical behavior, depending also on the loading mode (tension and compression). A compressive longitudinal loading regime resulted in the best load-bearing capacity for cortical bone, while tensile transverse loading provided significantly poorer results. The distinctive stress–strain curves obtained for tension and compression demonstrated various damage mechanisms associated with different loading modes. The variability of mechanical properties for different cortices was evaluated with two-way ANOVA analyses. Statistical significances were found among different quadrants for the Young's modulus. The results of microstructure analysis of the entire transverse cross section of a cortical bone also confirmed variations of volume fractions of constituents at microscopic level between anatomic quadrants:​ microstructure of the anterior quadrant was dominated by plexiform bone, whereas secondary osteons were prominent in the posterior quadrant. The effective Young's modulus predicted using the modified Voigt-Reuss-Hill averaging scheme accurately reproduced our experimental results, corroborating additionally a strong effect of random and heterogeneous microstructure on variation of mechanical properties in cortical bone.