Transfer RNAs (tRNAs) play an essential role in protein synthesis by linking the nucleic acid sequences of gene products to the amino acid sequences of proteins. Although only 32 tRNAs are needed to decode all 61 sense codons in the genetic code, there are > 400 functional tRNA genes in the humans. Adding to this diversity, there are many single nucleotide polymorphisms in tRNAs across our population, including anticodon variants that mistranslate the genetic code. In human genomes, we identified three alanine tRNA (tRNAAla) variants with non-synonymous anticodon mutations: tRNAAlaCGC G35T, tRNAAlaUGC G35A, and tRNAAlaAGC C36T. Since alanyl-tRNA synthetase (AlaRS) does not recognize the anticodon, we hypothesized that these human tRNAAla variants will mis-incorporate Ala at glutamate (Glu), valine (Val), and threonine (Thr) codons. We found that human cells expressing the naturally occurring tRNAAla variants were characterized by defects in protein production and cell growth. Using mass spectrometry, we confirmed and estimated Ala mis-incorporation levels at Glu (0.7%), Val (5%) and Thr (0.1%) codons. Although Ala mis-incorporation was higher at Val codons, cells mis-incorporating Ala at Glu codons had the most severe defect in protein production. Significant growth defects were observed in cells mistranslating Glu and Val codons, while the low level of mis-incorporation at Thr codons was well-tolerated in human cells. The data demonstrate the ability of natural human tRNAAla variants to generate mistranslation leading to a phenotypic response that depends on the nature of the amino acid replacement and the level of mis-incorporation.