To investigate the biosynthetic genes and the biological functions of carotenoids on the radioresistant mechanisms of the extremely radioresistant bacterium, Deinococcus radiodurans, the carotenoids of this bacterium were extracted with cold methanol and acetone, then were isolated and analyzed by LC-MS technique. Two colorless mutants knocking out of the phytoene synthase (crtB) and phytoene desaturase (crtI) genes were constructed by PCR and in vivo homologous recombination. The successful construction was confirmed by phenotype observation and carotenoids analysis by high performance liquid chromatography (HPLC). Comparative analysis of the sensitivities among the two colorless mutants and wild R1 showed that the two colorless mutants were more sensitive to ionizing radiation (IR) and ultraviolet (UV), indicating that the deletion of crtB and crtI genes made the mutants unable to biosynthesize the crucial intermediate lycopene and series of downstreaming carotenoids. The scavenging abilities of the two carotenoids were measured using the ultraviolet induction of bacteriophage λ (UIB) system and electron spin resonance (ESR) spin trapping technique in vivo and in vitro, respectively. The results showed that the main two carotenoids exhibited significant antioxidant capacities to superoxide anion (O₂^·) and hydroxyl radicals (·OH) which were two main types of free radicals produced during IR. This study has provided for the first time a new and direct evidence to explore the biosynthesis genes of carotenoids and their biological functions for the radioresistant mechanisms of D. radiodurans.