Protein kinase C epsilon (PKCvarepsilon), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCvarepsilon-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCvarepsilon signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCvarepsilon. Stat3 has two conserved amino-acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCvarepsilon interacts with Stat3alpha isoform, which has Ser727, and not with Stat3beta isoform, which lacks Ser727. PKCvarepsilon-Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that (1) PKCvarepsilon physically interacts with Stat3alpha isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231); (2) inhibition of PKCvarepsilon expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion; and (3) PKCvarepsilon mediates Stat3Ser727 phosphorylation through integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCvarepsilon-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.