AbstractPoly(ADP‐ribose) polymerase 1 (PARP1) plays a key role in genome stability by modulating DNA‐damage responses. Activated by DNA interruptions through ultraviolet (UV) exposure, PARylation is synthesized by PARP1 and serves as a survival mechanism for cancer and metabolic diseases. Several strategies including ROS and antimicrobial peptides (AMPs) function in host defenses, while the targeted tissue and mechanism under DNA damage are unknown. Here, we show that DNA damage induces responses specifically in the gut tissue. The knockdown of PARP1 reduces the activation of PARylation. Parp1 knockdown under DNA damage results in over‐accumulated ROS and secretion of AMPs through the regulation of Relish, a subunit of nuclear factor‐κB (NF‐κB). Double‐knockdown of Parp1 and Relish specifically in the gut inhibits AMP secretion. In conclusion, the host defense is achieved through ROS accumulation rather than the AMPs under DNA damage. In contrast, the knockdown of PARP1 exacerbates ROS accumulation to a harmful level. Under this circumstance, NF‐κb targeted AMP secretion is provoked for host defense. Microbiome and functional analysis provide evidence for the hazard of DNA damage and show variations in the metabolic pathways following Parp1 inhibition. Our findings suggest the notion that PARP1 inhibition contributes to ROS accumulation under DNA damage and its role in NF‐κb activation for host defense.