Myosin binding protein-C slow (sMyBP-C) comprises a family of accessory proteins in skeletal muscles that bind both myosin and actin filaments. Herein, we examined the role of sMyBP-C in adult skeletal muscles using in vivo gene transfer and clustered regularly interspaced short palindromic repeats technology to knock down all known sMyBP-C variants. Our findings, confirmed in two different skeletal muscles, demonstrated efficient knockdown (KD) of sMyBP-C (>70%) resulting in notably decreased levels of thick, but not thin, filament proteins ranging from ∼50% for slow and fast myosin to ∼20% for myomesin. Consistent with this, A bands were selectively distorted, and sarcomere length was significantly reduced. Contrary to earlier in vitro studies showing that addition of recombinant sMyBP-C slows down the formation of actomyosin crossbridges, our work demonstrates that KD of sMyBP-C in intact myofibers results in decreased contraction and relaxation kinetics under no-load conditions. Similarly, KD muscles develop markedly reduced twitch and tetanic force and contraction velocity. Taken together, our results show that sMyBP-C is essential for the regular organization and maintenance of myosin filaments into A bands and that its structural role precedes its ability to regulate actomyosin crossbridges.—Geist, J., Ward, C. W., Kontrogianni-Konstantopoulos, A. Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.