A synthetic EGF-like (EGFL) peptide (DdEGFL1), equivalent to the first EGFL domain in the extracellular matrix protein CyrA, has previously been shown to enhance random cell motility and cAMP-mediated chemotaxis in Dictyostelium discoideum. However the role of DdEGFL1 as a potential chemoattractant had not been addressed. In this study, a micropipette assay and an under-agarose migration assay showed that DdEGFL1 is not a chemoattractant for Dictyostelium cells. A radial bioassay was used to show that DdEGFL1 does not significantly enhance folate-mediated chemotaxis in contrast to its chemokinetic effect during chemotaxis toward cAMP. However, DdEGFL1 was able to rescue chemotaxis toward folate when the pathway was inhibited by pharmacological agents that inhibit known components of the signaling cascade (e.g. phosphatidylinositol 3-kinase, phospholipase A2, tyrosine kinases, and calmodulin). These data suggest that DdEGFL1 may activate a novel motility pathway that when coupled with folic acid receptor activation, can maintain the normal migratory response to folic acid in vegetative cells. Together, this data provides new insight into the function of EGFL repeats during Dictyostelium chemotaxis and the existence of a novel motility pathway regulated by EGFL peptides and/or repeats in this model organism.