The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca²⁺-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca²⁺ was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.