Ribonucleotide reductase (RNR) plays a critical role in catalyzing the biosynthesis and maintaining the intracellular concentration of 4 deoxyribonucleoside triphosphates (dNTPs). Unbalanced or deficient dNTP pools cause serious genotoxic consequences. Autophagy is the process by which cytoplasmic constituents are degraded in lysosomes to maintain cellular homeostasis and bioenergetics. However, the role of autophagy in regulating dNTP pools is not well understood. Herein, we reported that starvation- or rapamycin-induced autophagy was accompanied by a decrease in RNR activity and dNTP pools in human cancer cells. Furthermore, downregulation of the small subunit of RNR (RRM2) by siRNA or treatment with the RNR inhibitor hydroxyurea substantially induced autophagy. Conversely, cancer cells with abundant endogenous intracellular dNTPs or treated with dNTP precursors were less responsive to autophagy induction by rapamycin, suggesting that autophagy and dNTP pool levels are regulated through a negative feedback loop. Lastly, treatment with si-RRM2 caused an increase in MAP1LC3B, ATG5, BECN1, and ATG12 transcript abundance in xenografted Tu212 tumors in vivo. Together, our results revealed a previously unrecognized reciprocal regulation between dNTP pools and autophagy in cancer cells.