Phosphorus is an important macronutrient for plant growth, but often deficient in soil. To understand the molecular basis of the complex responses of potato (Solanum tuberosumL.) to phosphate (Pi) deficiency stress, the RNA-Seq approach was taken to identify genes responding to Pi starvation in potato roots. A total of 359 differentially expressed genes were identified, among which theSolanum tuberosumtranscription factor geneMYB44(StMYB44) was found to be down-regulated by Pi starvation.StMYB44was ubiquitously expressed in potato tissues and organs, and StMYB44 protein was exclusively localized in the nucleus. Overexpression ofStMYB44in potato resulted in lower accumulation of Pi in shoots. Transcriptomic analysis indicated that the abundance ofS. tuberosum PHOSPHATE1(StPHO1), a Pi transport-related gene, was reduced inStMYB44overexpression lines. In contrast, knock-out ofStMYB44by a CRISPR/Cas9 system failed to increase transcription ofStPHO1. Moreover, StMYB44 was found to interact in the nucleus with AtWRKY6, a known Arabidopsis transcription factor directly regulatingPHO1expression, and StWRKY6, indicating that StMYB44 could be a member of the regulatory complex controlling transcription ofStPHO1. Taken together, our study demonstrates thatStMYB44negatively regulates Pi transport in potato by suppressingStPHO1expression.