Persistent tyrosine phosphorylation of Stat3 and Stat5 is associated with oncogenic activity. Phosphorylation of the conserved tyrosine residue (pTyr) was long believed to be the only essential prerequisite to promote activation and nuclear translocation of Stat proteins. It has become evident, however, that post-translational protein modifications like serine phosphorylation, acetylation, glycosylation as well as protein splicing and processing constitute further regulatory mechanisms to modulate Stat transcriptional activity and to provide an additional layer of specificity to Jak-Stat signal transduction. Significantly, most vertebrate Stat proteins contain one conserved serine phosphorylation site within their transactivation domains. This phosphorylation motif is located within a P(M)SP sequence. Stat transcription factor activity is negatively influenced by mutation of the serine to alanine. Moreover, it was shown for both Stat3 and Stat5 that their capacity to transform cells was diminished. This review addresses recent advances in understanding the regulation and the biochemical and biological consequences of Stat serine phosphorylation. In particular, we discuss their role in persistently activated Stat proteins for cancer research.