Mesothelin (MSLN) overexpression in pancreatic cancer (PC) leads to enhanced cell survival/proliferation and tumor progression. After screening for a number of growth factors/cytokines, we found that the MSLN expression correlated closely with interleukin (IL)-6 in human PC specimens and cell lines. Stably overexpressing MSLN in different PC cell lines (MIA-MSLN and Panc1-MSLN) led to higher IL-6 production. Silencing MSLN by small interfering RNA (siRNA) significantly reduced IL-6 levels. Blocking the observed constitutive activation of nuclear factor-kappaB (NF-κB) with IKK inhibitor wedelolactone in MIA-MSLN cells also reduced IL-6. Silencing IL-6 by siRNA reduced cell proliferation, cell cycle progression and induced apoptosis with significant decrease of c-myc/bcl-2. Interestingly, recombinant IL-6-induced proliferation of MIA-MSLN cells but not MIA-V cells. Although messenger RNA/protein levels of IL-6R did not vary, soluble IL-6R (sIL-6R) was significantly elevated in MIA-MSLN and was reduced by treatment with the TACE/ADAM17 inhibitor TAPI-1, indicating intramembrane IL-6R cleavage and IL-6 trans-signaling may be operative in MIA-MSLN cells. Blocking the IL-6/sIL-6R axis using sIL-6R antibody abrogated basal proliferation/survival as well as recombinant human IL-6-induced cell proliferation. Our data suggest that MSLN-activated NF-κB induces elevated IL-6 expression, which acts as a growth factor to support PC cell survival/proliferation through a novel auto/paracrine IL-6/sIL-6R trans-signaling. In addition, using a panel of PC cells with varying MSLN/IL-6 expressions, we showed that MSLN/IL-6 axis is a major survival axis in PC supporting tumor cell growth under anchorage-dependent and independent conditions. The close correlation between MSLN and IL-6 provides a new rationale for combination therapy for effective control of MSLN-overexpressing PCs.