Ischemia and sepsis lead to endothelial cell damage resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase of F-actin along with a decrease in G-actin at 60 minutes. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of ADF/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild type Xenopus ADF/cofilin green fluorescent protein (XAC(wt)-GFP), GFP and the constitutively active and inactive isoforms, XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP depleted XAC(wt)-GFP and XAC(S3A)-GFP expressing cells, while increased actin stress fibers were observed in XAC(S3E)-GFP expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM Kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.