The spread of colistin-resistant bacteria is a serious threat to public health. As an alternative to traditional antibiotics, antimicrobial peptides (AMPs) show promise against multidrug resistance. In this study, we investigated the activity of the insect AMPTricoplusia nicecropin A (T. nicecropin) against colistin-resistant bacteria.T. nicecropin exhibited significant antibacterial and antibiofilm activities against colistin-resistantEscherichia coli(ColREC) with low cytotoxicity against mammalian cells in vitro. Results of permeabilization of the ColREC outer membrane as monitored through 1-N-phenylnaphthylamine uptake, scanning electron microscopy, lipopolysaccharide (LPS) neutralization, and LPS-binding interaction revealed thatT. nicecropin manifested antibacterial activity by targeting the outer membrane ofE. coliwith strong interaction with LPS.T. nicecropin specifically targeted toll-like receptor 4 (TLR4) and showed anti-inflammatory activities with a significant reduction of inflammatory cytokines in macrophages stimulated with either LPS or ColREC via blockade of TLR4-mediated inflammatory signaling. Moreover,T. nicecropin exhibited anti-septic effects in an LPS-induced endotoxemia mouse model, confirming its LPS-neutralizing activity, immunosuppressive effect, and recovery of organ damage in vivo. These findings demonstrate thatT. nicecropin exerts strong antimicrobial activities against ColREC and could serve as a foundation for the development of AMP therapeutics.