BACKGROUND Paraquat is used widely as one of the bipyridine herbicides, which generates reactive oxygen species to cause cell death. With a growing number of paraquat-resistant weeds, the mechanism of paraquat-resistance in plants remains unclear. This research verified the functions of a previously confirmed putative paraquat-resistant gene, EiKCS, from paraquat-resistant goosegrass by genetic engineering in a single overexpressing line in rice. RESULTS Overexpression of EiKCS improved paraquat resistance in transgenic rice (KCSox). Pre-applied (12 h) exogenous spermidine (1.5 mmol L-1), alleviated the injury of paraquat in rice. Paraquat induced injury in KCSox was 19.57%, which was lower than 32.22% injury it induced in wild-type (WT) rice. The paraquat-resistant mechanism was through the increased activity of antioxidant enzymes and the overproduction of endogenous polyamines. The spermine content in KCSox was more than 30 mu g mL(-1), while that in WT rice was less than 5 mu g mL(-1). Quantitative proteomics showed that beta-ketoacyl-coenzyme A (CoA) synthase (51.81 folds) encoded by the transgenic EiKCS gene promoted the synthesis of the proteins involved with the polyamine pathway. The synthesized putrescine was promoted by the arginine decarboxylase (ADC) pathway. The spermidine synthase I (1.10-fold) and three eceriferum cofactors (CERs) were responsive to the paraquat stress. We validated putrescine (C18H20N2O2) spermidine (C28H31N3O3), and spermine (C38H42N4O4) in this study. CONCLUSION EiKCS encoding beta-ketoacyl-CoA synthase from goosegrass has been shown as an ideal candidate gene for engineering genetically modified organism (GMO) crops, as its overexpression does not only bring paraquat-resistance, but also have potential benefits without decreasing yield and rice grain quality.