SUMMARYThe UV RESISTANCE LOCUS 8 (UVR8) photoreceptor mediates plant responses to Ultraviolet‐B (UV‐B) wavelengths. The UVR8 dimer dissociates into monomers following UV‐B photoreception, a process accompanied by conformational changes that facilitate interaction of UVR8 with proteins that initiate responses. However, the importance of particular amino acids in maintaining UVR8 conformation and modulating protein interactions is poorly understood. Here we examine the roles of cysteine amino acids C231 and C335 in UVR8 structure and function. UVR8C231S,C335S mutant protein forms dimers and monomerizes similarly to wild‐type UVR8. UVR8C231S,C335S interacts with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) in plants to initiate photomorphogenic responses to UV‐B, although the interaction is weaker when examined in yeast two‐hybrid assays. Similarly, the interaction of UVR8C231S,C335S with REPRESSOR OF UV‐B PHOTOMORPHOGENESIS (RUP) proteins is weaker in both plants and yeast compared with wild‐type UVR8. Re‐dimerization of UVR8 in plants, which is mediated by RUP proteins, occurs with reduced efficiency in UVR8C231S,C335S. Fluorescence resonance energy transfer analysis indicates that UVR8C231S,C335S has an altered conformation in plants, in that the N‐ and C‐termini appear closer together, which may explain the altered protein interactions.