The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Deltasms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Deltasms1 mutation.