Dysferlinopathy (LGMD2B, LGMDR1, Myoshi Myopathy) is a genetic muscle disease caused by mutations in the DYSF gene resulting in the absence of Dysferlin, a membrane protein involved in muscle membrane repair (membrane patch). Dysferlin deficiency compromises the sarcolemma integrity and leads to progressive muscle degeneration. Gene replacement therapy is a promising therapeutic strategy for dysferlinopathy. However, the dysferlin coding sequence (~6.2 kb) exceeds the packing capacity of a single AAV. To overcome this hurdle, dual AAV strategies based on homologous recombination of two vector genomes in myonuclei have been used to express the full-length Dysferlin in animal muscles. The requirement for delivering vector genomes from the two AAVs into the same nuclei in muscle fibers, as well as low efficiency of homologous recombination in post mitotic cells results in ineffective production of full length dysferlin with this approach. To address this, we used an alternative dual AAV strategy utilizing split protein inteins to express full-length Dysferlin. In this strategy, two segments of the dysferlin protein are expressed individually and the full length protein is generated by protein splicing in the muscle fiber cytoplasm. We tested multiple split inteins in vitro, and identified a lead dual AAV candidate containing a split intein that results in high efficiency of reconstitution and full length dysferlin protein production in myotubes. Systemic delivery of dual MyoAAVs for the lead candidate in Bla/J mice at low dose resulted in physiological or supraphysiological full-length dysferlin expression in multiple skeletal muscles. Immunofluorescence analysis showed widespread dysferlin expression across most muscle fibers, which provided protection against strain injury in Bla/J muscles. Our results provide evidence for the feasibility of using a dual AAV strategy utilizing split protein inteins for effective expression of full-length dysferlin protein in dystrophic muscle following a low-dose systemic MyoAAV administration.