Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.