Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells’ proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.