Motile cilia and flagella are whip-like subcellular organelles that bend actively to propel cells or move fluids. Normal motile functions of cilia and flagella play a critical role in many developmental and physiological processes, while ciliary dysfunction is associated with a number of ciliopathies. Efficient bending deformation of cilia and flagella depends on coordinated interactions between active forces from an array of motor proteins and passive mechanical resistance from the complex cytoskeletal structure (the axoneme). However, details of this coordination, especially axonemal mechanics, remain unclear. We investigated two major biophysical parameters, flexural rigidity and inter-doublet shear stiffness, of single flagella in the unicellular alga Chlamydomonas reinhardtii. Combining theoretical analysis with optical tweezers and counterbend experiments, we demonstrated that the apparent flexural rigidity of the axoneme depends on both the intrinsic flexural rigidity and the elastic inter-doublet shear stiffness. By comparing wild-type flagella with specific structural mutants, we found that the lack of nexin-dynein regulatory complexes (N-DRC) or dynein arms significantly reduces inter-doublet shear stiffness. The quantitative understanding of axonemal mechanics will ultimately lead to the development of novel diagnostic and therapeutic methods for cilia-related disorders.