Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog micro-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target micro-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the micro-dystrophin that restored dystrophin expression. MGNs also restored micro-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted micro-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.