The pro-peptide sequence of human parathyroid hormone (hPTH) is essential for the efficient translocation of the nascent polypeptide and the precise cleavage at residue +1 from the cleavage site. If residue +1 is not a serine, the bioactivity of hPTH decreases dramatically. In order to express the functional hPTH properly in a gene therapy model, we constructed three sets of eukaryotic expression vectors: one combining hPTH cDNA with its pre-sequence or its prepro-sequence; one combining hPTH cDNA with the insulin pre-sequence or with the insulin pre-sequence and the hPTH pro-sequence; one combining pro-insulin cDNA with the hPTH pre-sequence or prepro-sequence. Radioimmunoassay and reverse transcription/polymerase chain reaction showed that transgenes were expressed in the animal model. The hPTH expressed by the vectors with the pro-sequence of PTH improved bone mechanical properties and bone mineral content, whereas the expressed insulin decreased blood glucose. Our results demonstrated that the pro-sequence of hPTH was required for skeletal muscle cells to secrete functional hPTH into the circulation of our gene therapy model. However, the function of expressed insulin was not affected by the fusion of the hPTH pro-sequence. We concluded that pre-sequence, pro-sequence and mature hPTH acted as an integrated unit in skeletal muscle to guarantee the proper processing and release of functional hPTH into the circulation.