In order to study hypoglycemic effect after direct injection of a doxycyline-controlled insulin expression plasmid to the skeletal muscle of streptozotocin-induced diabetic mice, Balb/C mice were induced to diabetes. 300 μg of prTA-tet4-rhINS was injected into the muscles of mouse hind legs bilaterally and fed with drinking water containing different concentrations of doxycycline right after the injection. Tail blood glucose (random glucose level) was measured everyday with glucose oxidase method. Total RNA were isolated from muscles and liver tissues and RT-PCR was used to analyze the rhINS mRNA level and mouse β-actin mRNA was used as an internal control. After injection of the plasmid prTA-tet4-rhINS, the blood glucose in diabetes mice decreased markedly for an average of 10 mmol/L and 5 mmol/L at doxycycline concentration of 2 g/L and 0.5 g/L in the drinking water respectively. prTA-tet4-rhIN injected diabetic mice gained mass, and urine volume decreased. This hypoglycemic effect lasted for nearly two weeks. Furthermore, the hypoglycemic effect was closely related to the concentration of doxycycline in the drinking water in a dose-dependent manner. RT-PCR proved the expression of human proinsulin mRNA in the total muscle RNA. It can be concluded that a single tetracycline controlled recombinant pro-insulin gene expression plasmid could decrease the glucose levels of streptozotocin-induced diabetic mice dose-dependently by doxycycline in the drinking water. Muscle can be used as good target tissue for foreign insulin gene transfer and expression.