Background: There have been no comprehensive studies of changes in heart gene expression due to ethanol exposure. Therefore, we attempted to determine gene expression in cultured neonatal rat cardiomyocytes exposed to ethanol (0, 10, 50, 100 mM) for 24 h. Methods: The total RNA extract of beating cardiomyocytes was evaluated by DNA microarray analysis, and fold changes (FCs) in differential gene expression of ethanol-exposed cardiomyocytes were analyzed against the control using Ingenuity Pathway Analysis (IPA) software. Results: The 1,394 genes with an |FC| of ≥1.8 were uploaded to IPA. IPA predicted 23 canonical pathways working in the ethanol groups. Three canonical pathways related to ethanol degradation- "Ethanol Degradation IV", "Oxidative Ethanol Degradation III", and "Ethanol Degradation II" -were inhibited in the ethanol groups. IPA predicted "ethanol" as an upregulated upstream regulator of the network with 22 downstream members for the 100 mM ethanol group only. Three members (NTRK2, TGFB3, and TLR8) were activated in all groups. Certain cellular functions were predicted to be changed dose-dependently. "Myocarditis" was dose-dependently inhibited, whereas "Cell death of heart cells" was dose-dependently activated. Several functions were inhibited in 50 mM ethanol only, eg, "Failure of heart" was enhanced in 50 mM ethanol only. Certain functions were activated in 100 mM ethanol only. "Cardiac fibrosis" was not predicted in any ethanol group. Conclusions: IPA predicted that ethanol-induced activation or inhibition of canonical pathways and functions of cardiomyocyte depended on ethanol concentration, and 3 networks related to heart functions for cardiomyocytes exposed to 3 concentrations of ethanol.