The Sprouty (Spry) family of proteins has been identified as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Testicular protein kinase 1 (Tesk1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including early endosomes, leading to inhibition of its translocation to membrane ruffles upon growth factor stimulation. Tesk1 binding leads to a loss of inhibition of basic fibroblast growth factor (bFGF)- and FGF receptor1 (FGFR1)-induced ERK phosphorylation by Spry2, an effect that is independent of its kinase activity. Tesk1 abrogates the interaction of Spry2 with the adaptor protein Grb2 which is essential for its ERK inhibitory activity. It also inhibits the serine dephosphorylation of Spry2 upon bFGF and FGFR1 stimulation by impeding the binding of the catalytic subunit of the serine/threonine phosphatase PP2A to Spry2. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with Spry2 activation or function, highlighting the importance of Tesk1’s subcellular localization in this context. This regulation of Spry2 function by Tesk1 is reiterated in bFGF-induced neurite outgrowth in PC12 cells. However, Tesk1 does not interfere with Spry2’s interaction with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor (EGFR). By selectively modulating Spry2’s effects, Tesk1 may serve as a molecular determinant of the signaling outcome.