Wang et al. show that immune cell infiltration of tumours, as well as immune cell phenotype, oscillate in a circadian manner, which can be harnessed to optimize cancer immunotherapy. In a mouse model of melanoma, the numbers of tumour infiltrating leukocytes (TILs) peaked in the evening, and this was due to fluctuating levels of the adhesion molecule ICAM-1 on endothelial cells. Treatment with chimeric antigen receptor (CAR) T cells in the evening, as compared to the morning, induced greater tumour shrinkage. Moreover, surface expression of the checkpoint molecule PD1 on CD8+ T cells peaked in the evening, and in mouse models of melanoma and colon carcinoma, treatment with PD1-targeted antibodies showed an effect in the evening but not in the morning. Similar time-of-day differences in TIL number and phenotype were found in humans with melanoma, albeit phase-shifted, as expected for diurnal humans versus nocturnal mice. The implications of these results with regards to timing of immunotherapy will need to be tested in future clinical trials.