Accumulation of free fatty acids in skeletal muscle may at least in part, contribute to insulin resistance and mitochondrial dysfunction leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits Akt/Protein kinase B (Akt) activity in response to insulin. However, the mechanisms regulating palmitate-induced apoptosis in muscle have not been well studied. The purposes of this study were (i) to determine if palmitate-induced apoptosis in C2C12 myotubes, is dependent on Bax to Bcl-2 binding, and (ii) to determine if the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate treated myotubes. To determine if temporal Akt activity coincided with Bax to Bcl-2 binding, an immunoprecipitation (IP) assay was performed in myotubes after 2-hours of serum starvation followed by 10 minutes of serum reintroduction. The contribution of Bax to palmitate-induced apoptosis was determined by treating myotubes with Bax siRNA. Palmitate treatment increased apoptosis in C2C12 myotubes as shown by a 2-fold increase in DNA fragmentation, a ~5-fold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. Furthermore, there was a 4-fold reduction in Bax to Bcl-2 binding after palmitate treatment, which mirrored the reduction in pAkt ^Ser473 phosphorylation. Additionally, treatment of the C2C12 myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax mediated apoptosis in C2C12 myotubes and that this effect corresponds to reductions in Akt ^Ser473 phosphorylation.