Pigmented yeasts like Rhodotorula sp., Phaffia rhodozyma and Sporobolomyces sp.producedcarotenoid represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries, not only because they can act as vitamin A precursors, but also for their coloring, antioxidant and possible tumor-inhibiting activity. The carotenoid pigment producing yeast isolated from soil sample of Parambikulam Tiger reserve, India was identified as Rhodotorula mucilaginosa. In the present study antioxidant assays viz., DPPH scavenging and hydroxyl radical scavenging assays were carried out with yeast carotenoid pigment. The maximum antioxidation characteristics of carotenoid by DPPH and hydroxyl radical scavenging antioxidantassays (77.18 and 74.74 %) were achieved by pigmentation ofR. mucilaginosaat the concentration of 100 µg ml-1. The pigment fromR. mucilaginosa was separated by thin layer chromatography (TLC) yielding three major fractions, viz., yellow, orange and red fractions. These fractions were further purified through high performance liquid chromatography (HPLC) before subjecting to FT-IR spectral analysis for their structural elucidation. The carotenoid pigment from R. mucilaginosa resulted in three peaks at retention time of 3.35, 4.55 and 5.92 min respectively.