IRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnological applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest.