In temperate countries, the occurrence of cyanobacterial blooms threatens drinking water resources. Consequently, cyanotoxins are increasingly considered in water treatment, and their reactions with chlorine used to disinfect drinking water are particularly investigated. This study presents new elements for further understanding of cylindrospermopsin chlorination, through reactants and by-products monitoring, UV spectrum examination, and cytotoxicity assessment on human intestinal Caco-2 cells. On the one hand, the evolution of mixture UV spectrum indicated that cylindrospermopsin was quickly transformed at least into one intermediate by-product. While mass spectrometry experiments confirmed that cylindrospermopsin was almost totally transformed within 5min, chlorine was consumed up to 20min after the beginning of the reaction with a rate of 5 mol per mol of toxin. Then, LC-MS analysis gave rise to the formation of a third cylindrospermopsin by-product in addition to 5-chloro-cylindrospermopsin and cylindrospermopsic acid previously identified. Thanks to the accurate mass measurement provided by the LTQ-Orbitrap mass spectrometer, this new and stable chlorination by-product was assigned the chemical formula C(13)H(18)N(4)O(7)S. On the other hand, both of the mitochondrial and lysosomal activities measured on Caco-2 cells revealed that cylindrospermopsin chlorination significantly decreases mixture cytotoxicity.