Abstract Background The harmful consequences of in utero irradiation on learning and memory have been recognised but the molecular mechanisms behind the damage are still unknown. Results Using a mass spectrometry-based approach, we investigated the long-term changes in the global cortical and hippocampal proteome 6 months after 0.1, 0.5 and 1.0 Gy in utero X-ray irradiation delivered on embryonic day 11 in male C57Bl/6 J offspring. We noted alterations in several signalling pathways involved in cognition, the transcription factor cAMP response element-binding protein (CREB) playing a central role. Immunoblotting of CREB and phosphorylated CREB (Ser133) showed an altered expression profile at all doses in the hippocampus and at 0.5 and 1.0 Gy in the cortex. The greatest reduction in the phospho-CREB level was seen at 1.0 Gy in the hippocampus. It was accompanied by enhanced expression of postsynaptic density protein 95 (PSD95), suggesting effect on synaptic plasticity in neuronal dendrites. Conclusions As the CREB signalling pathway plays a crucial role in neuronal plasticity and long-term memory formation in the brain, the radiation-induced alterations of this pathway seen here are in good agreement with the cognitive dysfunction seen in in utero irradiated populations. These data contribute to a deeper biological understanding of molecular mechanisms behind the long-term damage induced by relatively low doses of ionising radiation during gestation.