L-plastin is a 70 kDa protein normally found in white blood cells, where it plays a pivotal role in the formation of actin-rich protrusions required for cell motility. Interestingly, many cancers hijack the white blood cell’s ability to move around and transform into metastatic cancers by upregulating L-plastin. Despite the potential clinical implications of this protein, the function and regulation of L-plastin remain enigmatic, and its full-length structure has yet to be fully characterized. By elucidating the regulatory mechanism of this proposed cancer biomarker, we may find a new approach to manipulate it and thereby reduce the motility of metastatic cancers. Regulation of the actin-bundling activity of L-plastin has been presumed to involve a structural change brought about by the binding of calcium to the N-terminal calcium-binding region (EF-hands). Intriguingly, in plants and yeast this region seems to be conserved, and yet incapable of sensing calcium. This implies another calcium-sensing mechanism may be involved in its regulation. To gain deeper insights into the regulation of L-plastin, we investigated the EF-hands to identify potential proteins involved in modulating its activity and elucidated the structures of the EF-hands of plant and yeast plastins using X-ray crystallography. Using nuclear magnetic resonance (NMR), we uncovered a calcium-sensing protein, calmodulin (CaM), involved in binding and potentially regulating L-plastin. The high-resolution studies of the EF-hands in plants and yeast suggest unique regulatory mechanisms that could enable plant and yeast plastins to function autonomously, independent of calcium. Identifying new mechanisms for actin-bundling can have a significant impact on the advancement of drug-development for the treatment of metastatic cancers.