Myocardial fibrosis is the endpoint pathology common to many cardiovascular disorders. We have previously shown that apocynin (APO), a naturally occurring NADPH oxidase inhibitor, significantly prevents the development of isoproterenol (ISO)-induced myocardial injury and fibrogenesis. The current study investigated the changes in microRNAs (miRNAs) and their potential implication in the cardioprotective effects of APO. Integrative analyses of whole-genome miRNA and gene expression profiles were first performed, revealing that altered expression of miRNAs likely contributed to dysregulated expression of genes associated with multiple interconnected fibrogenic signaling pathways. Importantly, APO treatment exhibited a broad impact on these signaling pathways, which could in part be mediated through miRNA-mediated gene expression regulation. The expression of differentially expressed miRNAs was further validated by real-time PCR analyses. Consistent with the data from miRNA array, compared to that from vehicle-treated normal controls, significantly decreased expression of miR-10b, miR-29c*, miR-30c-1*, miR-30e*, miR-148b, miR-181d, miR-218 and miR-3107* was observed in ISO-challenged vehicle-treated mouse hearts. In contrast, significantly increased expression of these miRNAs was observed in ISO-challenged APO-treated hearts compared to that from ISO-challenged vehicle-treated mice. Moreover, increased expression of miR-21 was observed as a result of ISO administration, which was significantly reduced by APO treatment. Altered protein levels of Col1, TIMP1, Rac2 and gp91phox were also validated. Lastly, APO treatment was shown to attenuate pre-established myocardial fibrosis induced by ISO. The results therefore demonstrated for the first time that complex changes in miRNA-mRNA interactome network are associated with the protective effects of APO against ISO-induced myocardial injury and fibrogenesis.