Cardiac hypertrophy, the adaptive response of the heart to overload, is a major risk factor for heart failure and sudden death. Estrogen (E2) and estrogen receptor beta (ERbeta) offer protection against hypertrophy and in the transition to heart failure. However, the underlying pathways remain incompletely defined. We employed a publicly available microarray dataset of female wild-type (WT) and ERbeta knockout (BERKO) mice subjected to pressure overload-induced hypertrophy to perform a systematic investigation of the mechanisms involved in the protection conferred by the E2/ERbeta axis. We show that considerably more genes were modulated in response to pressure overload in BERKO mice than in WT mice. The majority of the identified candidates in BERKO mice were induced, while those in WT mice were repressed. Pathway analysis revealed a similar pattern. This study is the first to demonstrate that the lack of ERbeta led to a significant increase of inflammatory pathways. Mitochondrial bioenergetics- and oxidative stress-related pathways were also modulated. In conclusion, ERbeta acquires the role of gatekeeper of the genomic response of the heart to pressure overload-induced hypertrophy. This may offer the molecular explanation for its cardioprotective role. We consider the present study to be a useful resource and that it will contribute to downstream functional analysis and to the characterization of pathways with previously unknown role in hypertrophy.