The actin cytoskeleton is a complex network controlled by a vast array of intricately regulated actin-binding proteins. Human plastins (PLS1， PLS2， and PLS3) are evolutionary conserved proteins that non-covalently crosslink actin filaments into tight bundles. Through stabilization of such bundles， plastins contribute， in an isoform-specific manner， to the formation of kidney and intestinal microvilli， inner ear stereocilia， immune synapses， endocytic patches， adhesion contacts， and invadosomes of immune and cancer cells. All plastins comprise an N-terminal Ca-binding regulatory headpiece domain followed by two actin-binding domains (ABD1 and ABD2). Actin bundling occurs due to simultaneous binding of both ABDs to separate actin filaments. Bundling is negatively regulated by Ca， but the mechanism of this inhibition remains unknown. In this study， we found that the bundling abilities of PLS1 and PLS2 were similarly sensitive to Ca (pCa ~6.4)， whereas PLS3 was less sensitive (pCa ~5.9). At the same time， all three isoforms bound to F-actin in a Ca-independent manner， suggesting that binding of only one of the ABDs is inhibited by Ca. Using limited proteolysis and mass spectrometry， we found that in the presence of Ca the EF-hands of human plastins bound to an immediately adjacent sequence homologous to canonical calmodulin-binding peptides. Furthermore， our data from differential centrifugation， F?rster resonance energy transfer， native electrophoresis， and chemical crosslinking suggest that Ca does not affect ABD1 but inhibits the ability of ABD2 to interact with actin. A structural mechanism of signal transmission from Ca to ABD2 through EF-hands remains to be established.