Nebulin is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. Nebulin gene defects are associated with congenital nemaline myopathy. The functional role of nebulin was investigated in gastrocnemius muscles from neonatal wild-type (WT) and nebulin-knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared to WT. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared to WT on postnatal day 1 and deteriorated by 92% on postnatal day 7, consistent with a functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of ten cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that nebulin is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of nebulin's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy.