The objective of this study was to determine the effects of ethanol injections on protein expression in the nucleus accumbens shell (ACB-sh) of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar (W) rats. Rats were injected for 5 consecutive days with either saline or 1 g/kg ethanol; 24 h after the last injection, rats were killed and brains obtained. Micro-punch samples of the ACB-sh were homogenized; extracted proteins were subjected to trypsin digestion and analyzed with a liquid chromatography-mass spectrometer procedure. Ethanol changed expression levels (1.15-fold or higher) of 128 proteins in NP rats, 22 proteins in P, and 28 proteins in W rats. Few of the changes observed with ethanol treatment for NP rats were observed for P and W rats. Many of the changes occurred in calcium-calmodulin signaling systems, G-protein signaling systems, synaptic structure and histones. Approximately half the changes observed in the ACB-sh of P rats were also observed for W rats. Overall, the results indicate a unique response to ethanol of the ACB-sh of NP rats compared to P and W rats; this unique response may reflect changes in neuronal function in the ACB-sh that could contribute to the low alcohol drinking behavior of the NP line.