Ste20-related proline/alanine-rich kinase (SPAK) plays a role in regulating many biological activities, and interacts with K-Cl co-transporter 3 (KCC3); however, the importance of SPAK for KCC3 function has not been demonstrated. Here, we investigated the role of SPAK in KCC3-regulated cell invasiveness and tumor formation. We show that induction of KCC3 expression triggers activation of the NF-κB and SPAK signaling cascades, leading to activation of p38 mitogen-activated protein kinase (MAPK) and matrix metalloproteinase-2 (MMP2). A small interference RNA-mediated reduction in SPAK protein levels suppressed the invasive ability and oncogenic potential of cervical cancer cells, and decreased tumor formation in mouse xenografts. A combination of experimental approaches, including RT-PCR and real-time RT-PCR, gelatin zymography, NF-κB luciferase activity and chromatin immunoprecipitation assays, showed that the induction of KCC3 over-expression increased MMP2 expression and augmented binding of NF-κB to its putative SPAK promoter binding site, suggesting that the SPAK/MMP2 axis is up-regulated by NF-κB. Pharmacological inhibition of NF-κB or MMP2 abrogated KCC3-triggered, SPAK-dependent cell invasiveness. Furthermore, p38 MAPK was identified as the upstream regulator of KCC3-dependent MMP2 activation. We conclude that SPAK may promote KCC3-mediated tumor aggressiveness via the NF-κB/p38 MAPK/MMP2 axis.