AbstractIn the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco‐PDT, in contrast to the other two cell lines (T47D and T98G). While 300 μm diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 μm) start to regrow following blue‐light PDT and exhibit high viability. Cerco‐PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (~590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco‐PDT. U87 spheroids also experienced a respiratory collapse following cerco‐PDT, but retained half their glycolytic activity.