The intricate interplay between viruses and hosts involves microRNAs (miRNAs) to regulate gene expression by targeting cellular/viral messenger RNAs (mRNAs). Mouse mammary tumour virus (MMTV), the aetiological agent of breast cancer and leukaemia/lymphomas in mice, provides an ideal model to explore how viral and host miRNAs interact to modulate virus replication and tumorigenesis. We previously reported dysregulation of host miRNAs in MMTV-infected mammary glands and MMTV-induced tumours, suggesting a direct interaction between MMTV and miRNAs. To explore this further, we systematically examined all potential interactions between host miRNAs and the MMTV genome using advanced prediction tools. Leveraging miRNA sequencing data from MMTV-expressing cells, we identified dysregulated miRNAs capable of targeting MMTV. Docking analysis validated the interaction of three dysregulated miRNAs with the MMTV genome, followed by confirmation with RNA immunoprecipitation assays. We further identified host targets of these miRNAs using mRNA sequencing data from MMTV-expressing cells. These findings should enhance our understanding of how MMTV replicates and interacts with the host to induce cancer in mice, a model important for cancer research. Given MMTV’s potential zoonosis and association with human breast cancer/lymphomas, if confirmed, our work could further lead to novel miRNA-based antivirals/therapeutics to prevent possible MMTV transmission and associated cancers in humans.