Human calcitonin (hCT) is a 32-amino acid hormone which plays important roles in the regulation of calcium metabolism, treatment of osteoporosis, inhibition of osteoclastic activity and Paget bone disease. Recombinant hCT has been previously produced at low concentrations in plant-based systems with biological activity. To investigate if hCT could be effectively produced and accumulate at high concentrations in a plant system, we established transgenic tobacco plant lines expressing an optimized synthetic gene construct. Several strategies were applied to increase production levels including a strong over expression promoter, modified codon usage and organelle targeting. An hCT expression construct with tobacco codon preference was synthesized and attached to a chloroplastic signal sequence from the Rubisco small subunit gene of Brassica napus L. The construct was created by designing specific primers, using Polymerase Chain Reaction (PCR) and overlapping extension methods. Agrobacterium-mediated transformation was used to establish transgenic tobacco plants. The transgenic plants were analyzed by molecular methods and hCT concentration was determined by quantitative EASIA. hCT was expressed in the cytoplasm and accumulated in the chloroplast with a 26% efficiency. This is the first report describing expression and chloroplast targeting of hCT in transgenic plants.