Numerous studies have demonstrated that Na⁺/H⁺exchanger isoform 1 (NHE1) is elevated in myocardial diseases and its effect is detrimental. To better understand the involvement of NHE1, we have previously studied cardiac-specific NHE1 transgenic mice and shown that these mice develop cardiac hypertrophy, interstitial fibrosis, and cardiac dysfunction. The purpose of current study was to identify microRNAs and their mRNA targets involved in NHE1-mediated cardiac injury. An unbiased high-throughput sequencing study was performed on both microRNAs and mRNAs. RNA sequencing showed that differentially expressed genes were enriched in hypertrophic cardiomyopathy pathway by Kyoto Encyclopedia of Genes and Genomes annotation in NHE1 transgenic hearts. These genes were classified as contraction defects (e.g., Myl2, Myh6, Mybpc3, and Actb), impaired intracellular Ca²⁺homeostasis (e.g., SERCA2a, Ryr2, Rcan1, and CaMKII delta), and signaling molecules for hypertrophic cardiomyopathy (e.g., Itga/b, IGF-1, Tgfb2/3, and Prkaa1/2). microRNA sequencing revealed that 15 microRNAs were differentially expressed (2-fold, P < 0.05). Six of them (miR-1, miR-208a-3p, miR-199a-5p, miR-21-5p, miR-146a-5p, and miR-30c-5p) were reported to be related to cardiac pathological functions. The integrative analysis of microRNA and RNA sequencing data identified several crucial microRNAs including miR-30c-5p, miR-199a-5p, miR-21-5p, and miR-34a-5p as well as 10 of their mRNA targets that may affect the heart via NFAT hypertrophy and cardiac hypertrophy signaling. Furthermore, important microRNAs and mRNA targets were validated by quantitative PCR. Our study comprehensively characterizes the expression patterns of microRNAs and mRNAs, establishes functional microRNA-mRNA pairs, elucidates the potential signaling pathways, and provides novel insights on the mechanisms underlying NHE1-medicated cardiac injury.