Tardigrades are captivating organisms known for their resilience in extreme environments, including ultra-high-dose radiation, but the underlying mechanisms of this resilience remain largely unknown. Using genome, transcriptome, and proteome analysis of
Hypsibius henanensis
sp. nov.
, we explored the molecular basis contributing to radiotolerance in this organism. A putatively horizontally transferred gene, DOPA dioxygenase 1 (
DODA1
), responds to radiation and confers radiotolerance by synthesizing betalains—a type of plant pigment with free radical–scavenging properties. A tardigrade-specific radiation-induced disordered protein, TRID1, facilitates DNA damage repair through a mechanism involving phase separation. Two mitochondrial respiratory chain complex assembly proteins, BCS1 and NDUFB8, accumulate to accelerate nicotinamide adenine dinucleotide (NAD
+
) regeneration for poly(adenosine diphosphate–ribosyl)ation (PARylation) and subsequent poly(adenosine diphosphate–ribose) polymerase 1 (PARP1)–mediated DNA damage repair. These three observations expand our understanding of mechanisms of tardigrade radiotolerance.