Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element-binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3beta (GSK3beta) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K(+) (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K(+) channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3beta inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3beta mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3beta inhibitors Li(+) and/or CHIR-99021, Li(+) increased IKACh, and Li(+) and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3beta activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3beta as a new therapeutic target for diabetic autonomic neuropathy.