Standard therapeutic management of insulin-dependent diabetes mellitus with intermediate-acting insulin poses a significant risk for iatrogenic hypoglycemia and associated hypoglycemia-associated autonomic failure. Gender differences in preservation of counterregulatory function during recurring hypoglycemia have been documented in the clinical setting. In rats, repeated induction of prolonged hypoglycemia by neutral protamine Hagadorn insulin (NPH) results in diminished neuronal transcriptional activation in several key metabolic loci in male, but not female rat brain, including the hindbrain dorsal vagal complex (DVC). Glucose is committed to glycolytic catabolism by hexokinase-mediated phosphorylation. The low-affinity, high K (m) hexokinase, glucokinase (GCK), monitors intracellular glucose levels in pancreatic beta cells, and is purported to fulfill a similar function in the CNS. GCK is expressed in the rat DVC, where mRNA is localized to neurons that exhibit electrophysiological sensitivity to glucose imbalance. The current study investigated the hypothesis that DVC GCK gene expression acclimates to RIIH in a gender-dependent manner. Quantitative real-time RT-PCR was used to evaluate GCK mRNA levels in microdissected DVC tissue obtained from male and female rats before or after one or serial doses of NPH. Basal DVC GCK transcripts were equal between ovariectomized (OVX) female rats implanted with estradiol benzoate (EB) or oil (O). Tissue mRNA levels were increased following a single NPH injection in both groups, but this response was greater in the presence of estrogen. Basal GCK gene expression was elevated by precedent insulin dosing in both O- and EB-implanted rats; however, transcripts were not modified relative to this adjusted baseline during subsequent NPH treatment in either group of females. While acute NPH treatment did not modify tissue transcript levels in orchidectomized (ORDX) or sham-ORDX rats, precedent NPH dosing increased basal GCK mRNA levels and further enhanced gene expression during ensuring hypoglycemia in sham males. These studies provide novel evidence for gender-dimorphic DVC GCK gene responses to single and serial intermediate-acting insulin administration. Further research is required to determine if and how stimulatory versus inhibitory transcriptional adaptation of this gene profile in male and female rats, respectively, impacts glucose-sensing functions in the DVC, and whether such adjustments may contribute to gender differences in magnitude of precedent hypoglycemic impairment of counterregulation.