In order to apply curcumin as a photosensitizer in photodynamic therapy (PDT) one needs a formulation that can solubilize and stabilize the compound. Pluronics® (Pluronic) are reported to both solubilize and stabilize curcumin against hydrolytic degradation. The aim of the present work was therefore to investigate the influence of Pluronic formulation on the photocytotoxicity of curcumin. Interactions between curcumin and Pluronics were investigated by fluorescence emission and absorption spectroscopy. Cell survival was measured with the MTT assay. The location of curcumin in the cells was investigated with fluorescence microscopy, and the cellular uptake was measured with fluorescence emission spectroscopy. Pluronics P123 and F127 in contrast to Pluronic P85 and PEG 400 may solubilize curcumin under non-cytotoxic conditions. An inverse relationship between the concentration of Pluronic and the photocytotoxicity of curcumin was observed. Curcumin could rapidly translocate across the cell membrane by passive diffusion. The fluorescence from curcumin in the cells (in the cytoplasm) after 1 hour of incubation was lowered by the presence of Pluronics in the formulation. However, the absolute amount of cell-bound curcumin after 1 hour of incubation was independent of the presence of Pluronics. Curcumin was bound more strongly to cells when incubated with formulations without Pluronics compared to cells incubated with curcumin formulations with Pluronics. Incubation of WiDr cells with curcumin for 6 hours resulted in lysosomal accumulation of curcumin independent of the presence of Pluronics. Lysosomally located curcumin could not be observed in HT1080 cells after 6 hours of incubation. The Pluronics P123 and F127 were found to be suitable for solubilizing and stabilizing curcumin, but inhibited photocytotoxic effects of curcumin unless the Pluronic concentration during treatment of the cells was less than 5–10× above the critical micellar concentration.