Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85α or p85β exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85α^−/− p55α^−/− p50α^−/− mice, where a decrease in p110α and p110β catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110α^+/− p110β^+/− mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110α^+/− p110β^+/− mice showed a ∼50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.