© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Utilizing the intrinsic mobility–strain relationship in semiconductors is critical for enabling strain engineering applications in high-performance flexible electronics. Here, measurements of Hall effect and Raman spectra of an organic semiconductor as a function of uniaxial mechanical strain are reported. This study reveals a very strong, anisotropic, and reversible modulation of the intrinsic (trap-free) charge carrier mobility of single-crystal rubrene transistors with strain, showing that the effective mobility of organic circuits can be enhanced by up to 100% with only 1% of compressive strain. Consistently, Raman spectroscopy reveals a systematic shift of the low-frequency Raman modes of rubrene to higher (lower) frequencies with compressive (tensile) strain, which is indicative of a reduction (enhancement) of thermal molecular disorder in the crystal with strain. This study lays the foundation of the strain engineering in organic electronics and advances the knowledge of the relationship between the carrier mobility, low-frequency vibrational modes, strain, and molecular disorder in organic semiconductors.