In this study, we utilized gene knockout and overexpression techniques to generate brewer’s yeast strains with either a deletion or overexpression of the fatty acyl-CoA oxidase (POX1) gene. The strains studied included the parental strain, the POX1 deletion strain, and the POX1 overexpression strain. These strains were exposed to iso-α acid from hops at a concentration of 300 mg/L, leading to the induction of a viable but non-culturable (VBNC) state. Our results indicated that the silencing of the POX1 gene rendered brewer’s yeast cells unable to withstand the high concentration of iso-α acid stress, ultimately leading to cell death. Conversely, the overexpression of POX1 accelerated the transition of yeast cells into the VBNC state compared to the parental strain. Furthermore, we evaluated the levels of reactive oxygen species (ROS), catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione reductase (GR) activity, and the mRNA expression of genes that regulate these enzymes (the stress-inducible yeast Mpv17 (SYM1) gene, CTA1, SOD1, and glutathione reductase (GLR1) gene) in brewer’s yeast cells at three distinct stages: normal, short-term stress, and VBNC states. Based on these findings, it can be inferred that the formation of the VBNC state in brewer’s yeast is associated with the response to oxidative stress.