AbstractPositron (β⁺) emitting radionuclides have been used for positron emission tomography (PET) imaging in diagnostic medicine since its development in the 1950s. Development of a fluorinated glucose analog, fluorodeoxyglucose, labelled with a β⁺ emitter fluorine-18 (¹⁸F-FDG), made it possible to image cellular targets with high glycolytic metabolism. These targets include cancer cells based on increased aerobic metabolism due to the Warburg effect, and thus, ¹⁸F-FDG is a staple in nuclear medicine clinics globally. However, due to its attention in the diagnostic setting, the therapeutic potential of β⁺ emitters have been overlooked in cancer medicine. Here we show the first in vitro evidence of β⁺ emitter cytotoxicity on prostate cancer cell line LNCaP C4-2B when treated with 20 Gy of ¹⁸F. Monte Carlo simulation revealed thermalized positrons (sub-keV) traversing DNA can be lethal due to highly localized energy deposition during the thermalization and annihilation processes. The computed single and double strand breakages were ~ 55% and 117% respectively, when compared to electrons at 400 eV. Our in vitro and in silico data imply an unexplored therapeutic potential for β⁺ emitters. These results may also have implications for emerging cancer theranostic strategies, where β⁺ emitting radionuclides could be utilized as a therapeutic as well as a diagnostic agent once the challenges in radiation safety and protection after patient administration of a radioactive compound are overcome.