Calmodulin (CaM) is a major cytoplasmic calcium receptor that performs multiple functions including cell motility. To investigate the mechanism of the regulation of CaM on cell morphology and motility, first we checked the distribution of CaM in the living cells using GFP-CaM as an indicator. We found that GFP-CaM showed a fiber-like distribution pattern in the cytosol of living Potorous tridactylis kidney (PtK2) cells but not in living HeLa cells. The endogenous CaM in heavily permeabilized HeLa was also found to display a fiber-like distribution pattern. Further examination showed that the distribution pattern of GFP-CaM was same as that of stress fibers, but not microtubules. Co-immunoprecipitation also showed that CaM can interact with actin directly or indirectly. The microinjection of trp peptide, a specific inhibitor of CaM, attenuated the polymerization of stress fibers and induced the alteration of cell morphology. A wound-healing assay and a single cell tracking experiment showed that CaM in PtK2 cells could increase cell motility. The data we have got from living cells suggested that CaM affect cell morphology and motility through binding to stress fibers and regulate f-actin polymerization.