Iodine-129 is present in controlled liquid radioactive waste routinely released in seawater by the ORANO nuclear fuel reprocessing plant in La Hague (Normandy, France). Brown algae are known for their exceptional ability to concentrate iodine from seawater. They also potentially emit volatile iodine compounds in response to various stresses, such as during emersion at low tide. For these reasons, brown seaweed is routinely collected for radioactivity monitoring in the marine environment (Fucus serratus and Laminaria digitata). Despite the high concentration ratio, the exact mechanism of iodine uptake is still unclear. Chemical imaging by laser desorption/ionization mass spectrometry provided evidence that iodine is stored by kelps as I−. In this study we investigate in vivo iodine uptake in kelps (L. digitata) with an emphasis on seawater iodine chemical speciation. Our results showed that kelp plantlets were able to take up iodine in the forms of both IO3− and I−. We also observed transient net efflux of I− back to seawater but no IO3− efflux. Since the seaweed stores I− but takes up both IO3− and I−, IO3− was likely to be converted into I− at some point in the plantlet. One major outcome of our experiments was the direct observation of the kelp-based biogenic conversion of seawater IO3− into I−. On the basis of both IO3− and I− uptakes by the seaweed, we propose new steps in the possible iodine concentration mechanism used by brown algae.