Experiments were performed in C57BL/6J male mice to determine the effects of acetylcholinesterase (AChE) inhibitor pyridostigmine bromide (PB) and stress on cardiovascular function, structure, and apoptosis. Mice were studied for seven days under the following conditions: Controls (osmotic minipump with saline), PB (10 mg/kg/day, minipumps), shaker stress (45 stressors/day, minipump with saline) and PB+Stress combination. AChE activity was significantly reduced in all PB-treated mice. PB caused no changes in 24-h mean arterial pressure (MAP) or heart rate (HR). Stress increased 24-h MAP on day 1 and 24-h HR on day 7 in both Stress and PB+Stress groups. A significant reduction in the aortic wall thickness/diameter ratio (P <0.05 vs. control) and slightly reduced relative heart weight were observed in the PB group. These effects were blunted by simultaneous stress exposure. Immunochemistry was used to stain for Bax and Bcl-2 (apoptosis markers). There was a four-fold increase in Bax/Bcl-2 ratio in the heart of PB and PB+Stress treated mice while an attenuation was observed in aortic endothelium. Results suggest that a relatively short-term continuous PB exposure may have adverse effects on the heart and blood vessels, independently of changes in MAP and HR.