Abstract <p><i>Candida utilis</i> (<i>C. utilis</i>) has been recognized as a safe organism by the USFDA and can be used as a feed additive. <i>C. utilis</i> with high protein production and high efficiency of ammonium sulfate utilization was screened using ¹²C⁶⁺ ion beam irradiation mutagenic technology. The utilization efficiency of ammonia nitrogen and the enzyme activity of the ammonia nitrogen assimilation pathway of three mutants were analyzed to identify the strains with more efficient ammonia nitrogen utilization for the production industry. Furthermore, we assessed the reasons for the enhanced ammonia nitrogen utilization capacity of three mutations by analyzing the cell phenotype, the nucleotide sequence of ammonium transporter-encoding (AMT) genes, and the expression level of these genes, to explore the molecular mechanisms underlying the enhanced ammonia nitrogen utilization capacity of the mutants. We found that the high-protein mutant <i>C. utilis</i> Y-3-2 had better ability to assimilate and utilize ammonia than the original strain and that the up-regulation of the AMT gene was critical to improving the utilization of ammonia nitrogen. Overall, our findings prove that ¹²C⁶⁺ beam radiation is effective in the production and utilization of <i>C. utilis</i> mutants, and provides important clues for us to further understand the molecular mechanism of enhanced ammonia utilization capacity of the mutants. Future studies will further explore the potential of these bacterial in other industrial applications and attempt to reveal more about the molecular mechanisms underlying the enhanced ammonia nitrogen utilization capacity.</p>