Methylotrophic yeast Komagataella phaffii is widely used in biotechnology for recombinant protein production. Due to the practical significance of these yeasts, it is extremely important to properly select cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on gene expression in K. phaffii at transcriptome level was investigated. It was demonstrated, that the response of K. phaffii cell to biotin deficiency strongly depends on the carbon source in the medium. In the media containing glycerol, biotin deficiency led to activation of the genes involved in biotin metabolism, glyoxylate cycle, and synthesis of acetyl-CoA in cytoplasm, as well as repression of the genes, involved in lipo- and gluconeogenesis. In the methanol-containing media, biotin deficiency primarily led to repression of the genes, involved in protein synthesis, and activation of cell response to oxidative stress.