Arginyltransferase1 (ATE1) is a conserved enzyme in eukaryotes mediating posttranslational arginylation, the addition of an extra arginine to an existing protein. In mammals, the dysregulations of the ATE1 gene ( ate1 ) is shown to be involved in cardiovascular abnormalities, cancer, and aging-related diseases. Although biochemical evidence suggested that arginylation may be involved in stress response and/or protein degradation, the physiological role of ATE1  in vivo  has never been systematically determined. This gap of knowledge leads to difficulties for interpreting the involvements of ATE1 in diseases pathogenesis. Since  ate1  is highly conserved between human and the unicellular organism  Schizosaccharomyces pombe  ( S. pombe ), we take advantage of the gene-knockout library of  S. pombe , to investigate the genetic interactions between  ate1  and other genes in a systematic and unbiased manner. By this approach, we found that  ate1  has a surprisingly small and focused impact size. Among the 3659 tested genes, which covers nearly 75% of the genome of  S. pombe , less than 5% of them displayed significant genetic interactions with  ate1 . Furthermore, these  ate1 -interacting partners can be grouped into a few discrete clustered categories based on their functions or their physical interactions. These categories include translation/transcription regulation, biosynthesis/metabolism of biomolecules (including histidine), cell morphology and cellular dynamics, response to oxidative or metabolic stress, ribosomal structure and function, and mitochondrial function. Unexpectedly, inconsistent to popular belief, very few genes in the global ubiquitination or degradation pathways showed interactions with  ate1 . Our results suggested that ATE1 specifically regulates a handful of cellular processes  in vivo , which will provide critical mechanistic leads for studying the involvements of ATE1 in normal physiologies as well as in diseased conditions.