2-Methylthio-N-6-isopentenyl modification of adenosine (ms(2)i(6)A) is an evolutionally conserved modification found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i(6)A) to ms(2)i(6)A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms 2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i(6)A by converting i(6)A to ms (2)i(6) A and protected GICs from excessive autophagy triggered by i(6)A. The elevated activity of CDK5RAP1 contributed to the amelioration of the tumor-suppressive effect of i(6)A and promoted GIC maintenance. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i(6)A and that GICs readily utilize this mechanism for survival