Aquaporin 5 (AQP5) is a water channel that regulates water transport, cell migration, and proliferation. Therefore, knowledge of its genetic regulation could be relevant to study these mechanisms. The AQP5 promoter region containing the AQP5-1364 A/C single-nucleotide polymorphism (SNP) might be an important regulatory region because the SNP is associated with the etiopathology of several diseases. The aim of this study was to identify a transcription factor that binds to this AQP5 promoter region and to investigate its potential impact upon AQP5 expression. In silico analysis revealed nuclear matrix protein 4 (NMP4) as a putative candidate. Electrophoretic mobility shift assays showed specific binding of NMP4 to the AQP5 promoter region of nt -1370 to nt -1329. Overexpression of NMP4 increased AQP5 promoter activity of the analyzed promoter constructs from nt -469 to nt -1979. Furthermore, an additional NMP4 binding site at position nt -592/nt -602 of the AQP5 promoter was identified. NMP4 overexpression increased AQP5 mRNA expression by 2.5-fold in HEK293 cells. Summarizing, we identified NMP4 as a novel transcriptional regulator of AQP5 expression, which binds to two AQP5 promoter regions. Both regions appear to impact AQP5 expression significantly.