In plants, low-dose of exogenous bacterial cyclic lipopeptides (CLPs) trigger transient membrane changes leading to activation of early and late defence responses. Here, a forward chemical genetics approach identifies colistin sulphate (CS) CLP as a novel plant defence inducer. CS uniquely triggers activation of the PATHOGENESIS-RELATED 1 (PR1) gene and resistance against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis thaliana (Arabidopsis) independently of the PR1 classical inducer, salicylic acid (SA) and the key SA-signalling protein, NON-EXPRESSOR OF PR1 (NPR1). Low bioactive concentration of CS does not trigger activation of early defence markers such as reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK). However, it strongly suppresses primary root length elongation. Structure activity relationship (SAR) assays and mode-of-action (MoA) studies show the acyl chain and activation of a ∼46 kDa p38-like kinase pathway to be crucial for CS' bioactivity. Selective pharmacological inhibition of the active p38-like kinase pathway by SB203580 reverses CS' effects on PR1 activation and root length suppression. Our results with CS as a chemical probe highlight the existence of a novel SA- and NPR1-independent branch of PR1 activation functioning via a membrane-sensitive p38-like kinase pathway.