Aquaglycero-aquaporins (agAQPs) are one of the water channel proteins located in the cell membrane that transport not only water but also some small solutes such as glycerol. Since agAQPs are involved in cancer proliferation and malignancy, it might be possible to utilize them as new targets for cancer molecular imaging. In this study, we investigated whether agAQPs can be specifically targeted by using [¹⁴C]-labeled glycerol ([¹⁴C]glycerol), which passes through agAQPs. In the in vitro experiments, comparing the cancer cell lines with different expression levels of AQP3 and AQP9, major agAQPs known to be expressed in cancers, and examining the effect of their inhibitors on these cells, the expression of AQP3 and AQP9 in cell lines was shown to be closely related to [¹⁴C]glycerol uptake. When [¹⁴C]glycerol was injected into tumor-bearing mice, Spearman’s rank coefficient analysis revealed that radioactivity levels in tumor and in plasma were mutually correlated only in tumors expressing agAQPs at a high level. These results indicate the possibility of using agAQPs as new targets to characterize cancer using radiolabeled glycerol as a molecular probe.