Cells balance glycolysis with respiration to support their energetic and biosynthetic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single-nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast, while most organisms possess isoforms with different activity. This variant reduced Pyk1 activity and glycolytic flux. Replacing the ‘low-activity’ pyk1 allele in the laboratory strain with the ‘high-activity’ allele was sufficient to increase fermentation and decrease respiration. This metabolic reprogramming triggered systems-level adaptations in the transcriptome and proteome, and in cellular traits, including increased growth and chronological lifespan, but decreased resistance to oxidative stress. Thus, low Pyk1 activity provides no growth advantage but stress tolerance, despite increased respiration. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate-kinase isoforms.