Oestradiol-17beta (E2) has been shown to exert anti-hypertrophic actions in the challenged and diseased heart. However, very little is known about the actions of E2 in the stress- and disease-free heart. Our initial aim was to identify and characterise structurally and molecularly the role of E2 in the healthy heart. Female C57Bl/6J mice were ovariectomised at the age of two months and were randomly assigned into groups feeding on either an E2-containing (n = 19) or soy-free (Ctrl; n = 19) diet for three months. Following this, the mice were sacrificed and hearts were collected for weight measurement. Left ventricles were analysed structurally by immunohistochemistry and molecularly by genome-wide expression profiling. E2 led to an increase in the heart weight (11%; P < 0.001) and the heart-to-body weight ratio (32%; P < 0.001) compared to Ctrl mice. Cardiomyocyte cross-sectional area revealed cardiomyocyte hypertrophy in E2 (n = 6) compared to Ctrl (n = 5) mice (32%; P = 0.004). Analysis of the left ventricular transcriptome identified 1059 probe sets (FDR-adjusted P ≤ 0.05) differentially expressed between E2 (n = 5) and Ctrl (n = 5). In particular, the expression of probe sets targeting the genes coding for β-catenin (Ctnnb1), cyclin D1 (Ccnd1), VEGF-A (Vegfa) and platelet/endothelial cell adhesion molecule 1 (Pecam1) was induced in the E2-treated group. Hypergeometric testing for Gene Ontology showed most genes to be associated with cell cycle, regulation of growth, cell and tissue development. Pathway analysis revealed 140 pathways (1000 permutations; P = 0.05) modulated between the two groups, such as the DNA replication and Wnt signalling pathways. To further confirm activation of Wnt/β-catenin signalling, we performed Western blotting. We identified that β-catenin translocated to the nucleus of mice treated with E2. Next, we tested the hypothesis that the hypertrophic effect of E2 is of the physiological type. To this extent, we identified that angiogenesis was increased with cardiac growth as determined by the microarray analysis and VEGF-A protein levels assessed by Western blotting. Furthermore, the embryonic gene programme was not activated and no fibrosis was observed in the E2-treated group. Based on our findings, we propose that the E2-induced hypertrophic effect is mediated through the regulation of the Wnt/β-catenin signalling pathway. Although at this point we have not characterised cardiac function, we expect this effect to be beneficial for the heart. Therefore, pharmacological modulation of the identified pathways might offer protection at early stages of cardiac disease.