The DUT gene encodes Deoxyuridine triphosphatase (dUTPase), which is involved in nucleotide metabolism. dUTPase prevents uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP. This study aimed to investigate the role of Dr-dut gene in the planarian Dugesia ryukyuensis by assessing the consequences of Dr-dut silencing on known phenomena, including regeneration following amputation and radiation damage. We functionally disrupted planarian Dr-dut mRNA by feeding RNAi-containing food to animals. Dr-dut RNAi resulted in the death of planarians in 28 days, and elevated double-stranded DNA breakage. Expression of the DNA damage response gene Dr-atm and the DNA repair genes Dr-rad51 and Dr-rad51c temporarily increased, and then decreased following the onset of feeding. When RNAi-treated planarians were amputated, both head and tail parts failed to regenerate, and the animals died in 25 and 29 days, respectively. Administration of 5-fluorouracil (5-FU) also resulted in death and DNA damage, and synergistically caused higher genotoxicity in planarian fed Dr-dut RNAi-containing food.