Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform which, in many cell types, is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of β-actin in fully differentiated muscle, we have performed a detailed characterization of wildtype and muscle-specific β-actin knockout (KO) mice. The effects of the β-actin KO were subtle. However, we confirmed the previously reported decline in running performance of β-actin KO mice compared to wildtype during repeated maximal running tests. We also found insulin-stimulated glucose transport into incubated muscles reduced in soleus but not in EDL of young adult mice. Contraction-stimulated glucose transport trended towards the same pattern, but the glucose transport phenotype disappeared in soleus muscles from mature adult mice. No genotype-related differences were found in body composition, glucose tolerance or by indirect calorimetry measurements. To evaluate β-actin mobility in mature muscle, we electroporated GFP-β-actin into FDB muscle fibers and measured FRAP. GFP-β-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, β-actin is not required for glucose transport regulation in mature mouse muscle under the majority of the tested conditions. Thus, our work reveals fundamental differences in the role of the cortical β-actin cytoskeleton in mature muscle compared to cell culture.