Organ transplant recipients must take immunosuppressive drugs daily to prevent transplant rejection. As a consequence of the systemic impact of these drugs, transplant patients suffer a greatly increased risk of cutaneous squamous cell carcinoma (SCC) development. The most prescribed immunosuppressive drug in these patients is the calcineurin inhibitor, tacrolimus. We proposed to develop a first in class, topically applied, small molecule antagonist of tacrolimus for the treatment of SCC in organ transplant recipients. Our lead compound, Q-2361, reversed the anti-proliferative effects of tacrolimus on both mouse and human T cells in vitro. To investigate whether Q-2361 impacted T cell activation and tumour regression in vivo, Q-2361 was injected into SCC tumours growing in tacrolimus-suppressed mice. Q-2361 treatment significantly reduced tumour growth and increased mouse survival compared to controls. Analysis of regressing tumours revealed that Q-2361 significantly increased both the activation of CD8 T cells within tumours, and their expression of effector molecules including IFN-gamma and TNF-alpha. Furthermore, when CD8 T cells were depleted in vivo, Q-2361 was no longer able to control tumour growth, indicating the mechanism of action of Q-2361 is via T cell activation. In summary, we demonstrate that the local application of Q-2361 mediates SCC regression in mice immunosuppressed with tacrolimus. Therefore, the topical application of Q-2361 warrants clinical evaluation for its capacity to prevent or reduce SCC growth in organ transplant recipients.