Pathological anxiety is paralleled by deficits in serotonergic and GABAergic neurotransmission in the amygdala. Conversely, anxiety disorders and depression may be reversed by brain-derived neurotrophic factor (BDNF). BDNF signaling involves Phosphatidylinositol 3-Kinase/ 3-phosphoinositide-dependent protein kinase 1 (PI3K/PDK1). We thus hypothesized that impaired function of PDK1 might be associated with increased anxiety and concomitant neurotransmitter changes. Here we used the hypomorphic PDK1hm mouse to investigate anxiety behavior in different settings: PDK1hm mice differed from Wt littermates PDK1WT in several behavioral measures related to anxiety and exploration, namely in the open field, dark-light box, O-maze and startle response. Further we analyzed the brain substrate underlying this phenotype and found significantly decreased GABA, taurine and serotonin concentrations in the amygdala and olfactory bulb of PDK1hm mice, while BDNF and nerve growth factor (NGF) concentrations were not significantly different between PDK1hm and PDK1WT mice. These results suggest that impaired PI3K signaling in the PDK1hm mouse reduces concentrations of GABA and serotonin in anxiety related brain regions and can serve as a molecular substrate for behavior indicative for anxious and depressive-like mood states.