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Epigenetic therapy using DNA methyltransferase (DNMT) inhibitors, has attracted a great interest for the treatment of solid tumours including colorectal cancer (CRC). Decitabine (DAC), a DNMT inhibitor, has been FDA-approved for the treatment of haematological malignancies. However, the therapeutic clinical outcome of DAC in solid tumours is limited by its short plasma half-life and degradation by cytidine deaminase, whilst myelosuppression can be induced at high doses. To enhance DAC delivery and improve its therapeutic potential in CRC in vitro and/or in vivo, free DAC delivery combined with ultrasound (US)-mediated microbubble (MB) destruction (UMMD) was tested. In addition, encapsulation of DAC within liposomes was performed. Drug-loaded liposomes can be conjugated to US-triggered MBs, which can be targeted to the vascular endothelial growth factor receptor 2 (VEGFR2) of the tumour endothelial cells. End-point assays using aberrantly-methylated genes that would respond to DAC treatment were identified including the tumour suppressor genes, CDO1 and SPARC. These were tested as potential epigenetic biomarkers of response in human CRC cell lines in vitro and in vivo. In vitro treatment with low dose DAC liposomes induced higher protein levels of SPARC in SW620 metastatic CRC (mCRC) cells compared to DAC in solution. Free DAC treatment followed by UMMD resulted in significantly higher SPARC protein levels in SW620 cells compared to DAC with US or DAC alone in vitro. CDO1 protein levels were increased in response to DAC with US with or without MBs compared to DAC alone in HCT116 cells. Free DAC delivery combined with UMMD reduced the tumour growth and tumour mass of SW620 human CRC xenografts compared to DAC with US. This was associated with significant epigenetic reactivation of SPARC. Upregulation of MAGEA3/6, a cancer-testis antigen, was induced in both cases compared to the vehicle control. It can be concluded that systemic DAC treatment combined with UMMD can enhance the epigenetic potential of DAC in a preclinical model of CRC in vivo.