In the present work, a novel cordycepic pigment was successfully isolated and identified from Cordyceps militaris, as well as named as cordycepene (C14H17N1O4), according to the long unsaturated conjugated polyene structural characteristic. Cordycepene is sensitive to light, high temperature (≥ 60 °C), and acidic condition (pH ≤ 3), but possesses high stability against metal ions, and under alkaline and neutral conditions. Cordycepene shows a comparable DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging activity at higher concentration (≥ 2 mg/mL) to vitamin C. Cordycepene promotes the growth of HSF (human skin fibroblast cell) after incubation for 72 h, and has an ability to repair the UV light-treated HSF cells. In addition, cordycepene increases the antioxidant activity (SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase) and decreases MDA (malondialdehyde) level, indicating that cordycepene inhibits the photochemical senescence of HSF by enhancing the antioxidant defense system. The discovery of cordycepene can provide a basis for research on light incubation and the accumulation of yellow pigment (carotenoids) from C. militaris.