PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1−/− mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107–143, whereas the other one encompasses amino acids 227–263 and displays repressor activity. Using an immunoprecipitation–MS approach, two phosphorylation sites were identified, Ser119 and Ser238. Phosphorylation at Ser119 is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser119 phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.