Type 1 diabetic patients depend dramatically on insulin replacement therapy, which involves the administration of intermediate- or long-acting insulin, together with short-acting insulin to mimic physiological insulin profiles. However, the delayed-action preparations available are not generally able to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stably transfected with a chimeric gene obtained by linking the myosin-light chain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm). When differentiated, C2C12Insm myotube cells expressed high levels of insulin mRNA and protein, whereas no insulin was detected in myoblast cells. HPLC fractionation of culture medium and cell extracts from differentiated C2C12Insm cells revealed that about 90% of the proinsulin was processed to mature insulin. In addition, these cells released significant levels (about 100 μU/106 cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the differentiated C2C12Insm cells and was able to block the expression of the endogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepatoma cells. Furthermore, when C2C12Insm myoblast cells were transplanted into diabetic mice an increase in insulinemia and a decrease in hyperglycemia were observed. Thus, our results suggest that the use of engineered myotube cells continuously secreting a defined level of insulin might be a useful approach to improve the efficacy of insulin injection treatment.