Sclerotinia sclerotiorum differentiates by forming terminal-type sclerotia. We found that the fungus produces β-carotene in low and high levels during its undifferentiated and differentiated stages, respectively, and at low (in dark) and high (in light) oxidative growth conditions, respectively. In contrast, a sclerotium non-producing strain of S. sclerotiorum produces very low amounts of β-carotene in constant levels throughout its entire growth period, irrespective of oxidative stress degree of growth conditions. When β-carotene was added to the growth medium of the sclerotium producing strain, it caused a concentration-dependent inhibition of sclerotia formation. We propose that β-carotene inhibits sclerotial differentiation in S. sclerotiorum by lowering oxidative stress via its antioxidant scavenging properties.