Astrocytes are supportive cells, necessary for ensure optimal environment for neural cells functioning. They are involved in extracellular K+ level regulation and neurotransmitters removal. They are also dependent for myelination and synapses formation. They may make a contribution in signal propagation in the central nervous system, for example, through Ca2+ signaling. With the use of neonatal pure astrocyte cell culture, we investigated changes in astrocyte's proteomes under the influence of morphine. We found 10 major proteins, which show different expression between physiological cell culture and morphine treatment. With 2D gel electrophoresis and nanoLC-ESI-MS/MS, we identified proteins and characterized their potential role in morphine dependence. Observed differences were also confirmed by Western blotting. Our data suggests a role for astrocytes in the formation of the morphine dependence at the molecular level. This finding may support interpretation of causes of morphine dependence formation based only on behavioral data.