Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific protein kinase C (PKC) substrate, mediates PKC signaling through its phosphorylation and subsequent modification of its association with filamentous actin (F-actin) and calmodulin (CaM). PKC has long been implicated in cell proliferation, and recent studies have suggested that MARCKS may function as a cell growth suppressor. Therefore, in the present study, we investigated MARCKS protein expression, distribution, and phosphorylation in preconfluent and confluent bovine pulmonary microvascular endothelial cells (BPMEC) in the presence or absence of the vascular endothelial growth factor (VEGF). In addition, we examined functional alterations of MARCKS in these cells by studying the association of MARCKS with F-actin and CaM-dependent myosin light chain (MLC) phosphorylation. Our results indicate that MARCKS protein is downregulated during BPMEC proliferation. Decreased MARCKS association with F-actin, increased actin polymerization, and CaM-dependent MLC phosphorylation appear to mediate cell shape changes and motility during BPMEC growth. In contrast, VEGF stimulated MARCKS phosphorylation without alteration of protein expression during BPMEC proliferation, which may result in reduced interaction between MARCKS and actin or CaM, leading to actin reorganization and MLC phosphorylation. Our data suggest a regulatory role of MARCKS during endothelial cell proliferation.