Dystrophin is a critical muscle cell structural protein which when deficient results in Duchenne muscular dystrophy. Recently miniature versions of the dystrophin gene have been constructed that ameliorate the pathology in mouse models. To characterize mini-dystrophin's incorporation into the dystrophin protein complex in living cells, two fusion proteins were constructed where mini-dystrophin is fused to the N- or C-terminus of an enhanced green fluorescent protein reporter gene. Both fusion proteins correctly localize at the plasma membrane in vitro and in vivo. Live cell microscopy establishes that mini-dystrophin translocates directly to the PM of differentiating muscle cells, within 4 h of expression. Latrunculin A treatment, actin and β-dystroglycan binding domain deletion constructs, and co-immunoprecipitation assays demonstrate that mini-dystrophin is firmly anchored to the sarcolemma primarily through its connections to β-dystroglycan, mimicking effects seen with wild type dystrophin. Furthermore, point mutations made within the putative β-dystroglycan anchoring ZZ domain of mini-dystrophin result in an ablation of β-dystroglycan binding and a nuclear translocation of the protein. These results demonstrate that mini-dystrophin is efficiently bound and incorporated into the dystrophin protein complex, via β-dystroglycan in living cells, similarly to the full length dystrophin protein.