In addition to participating in dosage compensation, the MLE helicase in D. melanogaster performs many functions in the regulation of gene expression, as does its human ortholog DHX9. Many of these functions are evolutionarily conserved and poorly explored. MLE has previously been shown to be involved in the regulation of inducible transcription of the ftz-f1 gene encoding the evolutionarily conserved nuclear receptor NR5A3. The ftz-f1 gene also encodes a constitutive transcript synthesized from an alternative promoter. The present work is devoted to the investigation of the role of MLE in the regulation of constitutive transcription of the ftz-f1 gene. This work shows that in S2 cell culture, MLE binds to the constitutive promoter and controls both inducible and constitutive transcription of the ftz-f1 gene. A novel MLE-binding cis- regulatory element of the ftz-f1 gene, enhancer 663, was identified. Using chromosome conformation capture technique the interaction of enhancer 663 with constitutive and inducible promoters of ftz-f1 gene in S2 cell culture was demonstrated. Examination of enhancer 663 histone H3 acetylation showed that it is involved in the activity of both promoters. Knockdown of MLE in S2 cell culture causes an increase in constitutive transcription. The effect of MLE on transcription beyond dosage compensation in vivo at the adult stage was shown for the first time. It was shown that at the adult stage MLE binds to both inducible and constitutive promoters and to enhancer 663. Mutation in the mle gene leads to increased expression of both transcripts of the ftz-f1 gene in females. The data obtained are important for understanding and further study of the evolutionarily conserved functions of MLE and its human ortholog DHX9.