Aims Enhancement of the tolerance of Saccharomyces cerevisiae to monoterpenes has the potential to improve the de novo biosynthesis of these chemicals as well as the efficient utilization of monoterpene-containing citrus waste. The aims of the current work are to demonstrate the mechanisms by which ergosterol, an important component of cell membranes, protects S. cerevisiae from d-limonene stress and to provide some useful information for further metabolic engineering of the yeast. Methods and Results Saccharomyces cerevisiae cells were treated with a sublethal dose of d-limonene for 2 h, and then ergosterol was added to investigate the physiological responses of S. cerevisiae. In d-limonene-treated cells, the membrane fluidity, permeability and saturated fatty acid ratio increased, whereas the intracellular ergosterol concentration decreased sharply. Addition of ergosterol restored membrane and intracellular ergosterol to normal levels. Exogenous ergosterol triggered nearly all of the genes that encode the biosynthesis of ergosterol. Conclusions In S. cerevisiae, the cell membrane is the target of d-limonene. Intracellular ergosterol availability is correlated with the d-limonene tolerance of the cells. Significance and Impact of the Study The results indicate that modification of the ergosterol biosynthesis pathway could be a promising strategy for constructing a robust yeast strain with enhanced tolerance.