Updated project metadata. Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here, we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential tRNA modification enzymes in GSCs. YRDC catalyses the formation of N6-threonylcarbamoyladenosine (t6A) on ANN-decoding tRNAs (A denotes adenosine and N denotes any nucleotide). Targeting YRDC reduced t6A formation, suppressed global translation, and inhibited tumour growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t6A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon-bias. Dietary threonine restriction (TR) reduced tumour t6A formation, slowed xenograft growth, and augmented anti-tumour efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.