This study demonstrates that UV radiation (UVR) reduces the photoprotective capacity of the diatom Phaeodactylum tricornutum by affecting xanthophyll cycle (XC) activity. The short-term reduction of photosystem II (PSII) maximum efficiency of charge separation (Fv/Fm) in cells exposed to UVR could be explained mainly by a reduced photoprotective capacity under this condition. Phaeodactylum tricornutum cells acclimated to two different photosynthetically active radiation (PAR) intensities, high light (HL, 200 μmol quanta m−2 s−1) and low light (LL, 50 μmol quanta m−2 s−1), were exposed to saturating irradiance (1100 μmol quanta m−2 s−1) in the presence (PAR + UVR) and absence of UVR (PAR). HL cells exhibited a greater reduction in Fv/Fm in PAR + UVR when compared with the PAR treatment that was related to a reduction in the de-epoxidation of XC pigments. In contrast, in LL cells, UVR did not considerably affect XC de-epoxidation even though the reduction in Fv/Fm was greater than in HL cells. The negative effect of UVR on photoprotection was more pronounced in HL cells because they synthesized more XC pigments than LL cells. This was confirmed when XC activity was blocked with dithiothreitol and when PSII repair was inhibited with chloramphenicol (CAP). The differential reduction of Fv/Fm between PAR + UVR and PAR treatments disappeared when XC was blocked in HL cells. A higher reduction and an incomplete recovery of Fv/Fm were observed in cells incubated with CAP in the presence of UVR. Such responses confirm that UVR had a negative effect on photoprotective mechanisms causing an enhancement of damage by PAR, especially in HL-acclimated cells in which heat dissipation is important for PSII regulation.