sef (similar expression to fgf genes) was recently identified as a negative regulator of fibroblast growth factor (FGF) signaling in zebrafish, chicken, mouse and human. By repressing events upstream and/or downstream Ras, Sef inhibits FGF-induced ERK activation and cell proliferation. Here we report that Sef-S, an alternative splice isoform of Sef, lacks a signal peptide and is localized in cytosol. Sef-S inhibits FGF-induced NIH3T3 cell proliferation, a similar function to Sef. However, Sef-S represses neither the intensity nor the duration of ERK activation. Moreover, Sef-S does not inhibit Elk1-dependent transcription. Our study revealed that the signal peptide is critical for the different activities between Sef and Sef-S in FGF-Ras-MAPK signaling cascades. Furthermore, we observed that Sef-S associated with FGFR2 in a co-immunoprecipitated complex. These results indicate that Sef-S inhibits FGF-induced NIH3T3 cell proliferation via an ERK-independent mechanism and therefore suggest that alternative splice licenses sef gene to inhibit cell proliferation via multiple signaling pathways.