The protein translational system, including transfer RNAs (tRNAs) and several categories of enzymes, plays a key role in regulating cell proliferation. Translation dysregulation also contributes to cancer development, though relatively little is known about the changes that occur to the translational system in cancer. Here, we present global analyses of tRNAs and three categories of enzymes involved in translational regulation in ~10,000 cancer patients across 31 cancer types from The Cancer Genome Atlas. By analyzing the expression levels of tRNAs at the gene, codon, and amino acid levels, we identified unequal alterations in tRNA expression, likely due to the uneven distribution of tRNAs decoding different codons. We find that overexpression of tRNAs recognizing codons with a low observed-over-expected ratio may overcome the translational bottleneck in tumorigenesis. We further observed overall overexpression and amplification of tRNA modification enzymes, aminoacyl-tRNA synthetases, and translation factors, which may play synergistic roles with overexpression of tRNAs to activate the translational systems across multiple cancer types.