Chronic acidosis is a stimulus for proteolysis in muscle in vivo, but the mechanism of this response is unknown. We tested the hypothesis that acidosis or TNF-alpha, a cytokine whose production increases in acidosis, regulates proteolysis by inhibiting insulin signaling through phosphoinositide 3-kinase (PI3K). In cultured L6 myotubes, acidified (pH 7.1) media did not accelerate the basal protein degradation rate, but it inhibited insulin's ability to suppress proteolysis. Insulin receptor substrate-1 (IRS-1)-associated PI3K activity was not altered in cells acidified for 10 min but was strongly inhibited in cells incubated at pH 7.1 for 24 h. Phosphorylation of Akt was also suppressed by acidification for 24 h. Acidification did not induce changes in IRS-1 abundance, insulin-stimulated IRS-1 tyrosine phosphorylation, or the amount of PI3K p85 regulatory subunit. In contrast to acidification, TNF-alpha suppressed proteolysis in the presence or absence of insulin but had no effect on IRS-1-associated PI3K activity. To establish that the PI3K pathway can regulate protein degradation in muscle, we measured proteolysis in cells after inhibition of PI3K activity with LY-294002 or infection with an adenovirus encoding a dominant negative PI3K p85alpha-subunit. Both approaches inhibited insulin-induced suppression of proteolysis to a degree similar to that seen with acidification. We conclude that acidosis accelerates protein degradation by impairing insulin signaling through PI3K in muscle cells.