BACKGROUND: Recent studies on colour discrimination suggest that experience is an important factor in how a visual system processes spectral signals. In insects it has been shown that differential conditioning is important for processing fine colour discriminations. However, the visual system of many insects, including the honeybee, has a complex set of neural pathways, in which input from the long wavelength sensitive ( green ) photoreceptor may be processed either as an independent achromatic signal or as part of a trichromatic opponent-colour system. Thus, a potential confound of colour learning in insects is the possibility that modulation of the green photoreceptor could underlie observations. METHODOLOGY/PRINCIPAL FINDINGS: We tested honeybee vision using light emitting diodes centered on 414 and 424 nm wavelengths, which limit activation to the short-wavelength-sensitive ( UV ) and medium-wavelength-sensitive ( blue ) photoreceptors. The absolute irradiance spectra of stimuli was measured and modelled at both receptor and colour processing levels, and stimuli were then presented to the bees in a Y-maze at a large visual angle (26 degrees ), to ensure chromatic processing. Sixteen bees were trained over 50 trials, using either appetitive differential conditioning (N = 8), or aversive-appetitive differential conditioning (N = 8). In both cases the bees slowly learned to discriminate between the target and distractor with significantly better accuracy than would be expected by chance. Control experiments confirmed that changing stimulus intensity in transfers tests does not significantly affect bee performance, and it was possible to replicate previous findings that bees do not learn similar colour stimuli with absolute conditioning. CONCLUSION: Our data indicate that honeybee colour vision can be tuned to relatively small spectral differences, independent of green photoreceptor contrast and brightness cues. We thus show that colour vision is at least partly experience dependent, and behavioural plasticity plays an important role in how bees exploit colour information.