Xanthophyllomyces dendrorhoussynthesizes astaxanthin, a carotenoid used in aquaculture. Astaxanthin is synthesized from metabolites of the mevalonate pathway, which are also precursors for sterols biosynthesis. The interruption of theCYP61gene, which is involved in the synthesis of ergosterol (mutant CBS.cyp61(-)), resulted in a phenotype that overproduces carotenoids due to the activation of the SREBP pathway. In this work, we constructed other mutants of ergosterol biosynthesis in this yeast to evaluate whether they have the same phenotype as mutant CBS.cyp61(-). By bioinformatic analysis, theERG3andERG4genes ofX. dendrorhouswere identified, and each gene was deleted in the wild-type strain. Mutants CBS.Delta erg3and CBS.Delta erg4did not produce ergosterol; CBS.Delta erg3primarily accumulated episterol, and CBS.Delta erg4primarily accumulated ergosta-5,7,22,24(28)-tetraenol. The transcription levels of theHMGSgene of the mevalonate pathway were evaluated by RT-qPCR, which showed a slight increase in CBS.Delta erg4, but the transcription levels were still 10-fold lower than in strain CBS.cyp61(-). Both CBS.Delta erg3and CBS.Delta erg4did not overproduce carotenoids, even though they do not produce ergosterol. Thus, the results of this study indicate that the absence of ergosterol does not activate the SREBP pathway inX. dendrorhous, but rather it depends on other alterations in sterol composition.