Significance Actin filaments are the fundamental building blocks for cellular motility in muscle cells. However, the regulation of actin filaments in sarcomeres is poorly understood. Lmod is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability lead to lethal nemaline myopathy. Here we report the crystal structure of actin–Lmod2 complex and complementary functional studies. Our data collectively unraveled the mechanisms of Lmod’s essential functions as an actin nucleator and filament length regulator, thus providing a solid foundation for understanding the molecular basis of disease-causing mutations and malfunctions of Lmod that often are life threatening. Most importantly, our results suggest a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators.