<p>Promoters able to respond to moderate alkalinization in the industrially relevant yeast <i>Komagataella phaffii</i> have been proposed as a tool for the design of novel methanol-free platforms for heterologous protein expression. However, the molecular bases of such response are unknown. We identify here the transcription factors Crz1 and Rim101 in <i>K. phaffii</i> and use CRISPR/Cas9 techniques to create single and double mutant strains. While <i>crz1</i> cells are strongly sensitive to Ca²⁺ ions, mildly sensitive to alkaline pH and hypertolerant to SDS, <i>rim101</i> cells are markedly hypersensitive to Na⁺ and Li⁺ ions, to alkaline pH and to SDS. RNA-seq analysis revealed that mutation of <i>RIM101</i> affects 55% of alkaline pH-responsive genes, whereas lack of Crz1 alters only 11% of these. Thirty-eight genes are co-regulated by both Crz1 and Rim101. The <i>PMC1</i> vacuolar Ca²⁺-ATPase is regulated by Crz1, whereas the <i>ENA2</i> Na⁺-ATPase, the <i>PHO89</i> and <i>PHO84</i> phosphate transporters, and diverse genes involved in iron homeostasis are Rim101-dependent. These effects were confirmed by proteomic analyses after 3&#xa0;h of alkalinization. Therefore, Rim101 appears as a major player in the transcriptional response to alkalinization and, together with Crz1, they account for nearly 60% of the alkaline pH transcriptional response in <i>K. phaffii</i>.</p>