Weeds are a major biotic constraint that can cause dramatic crop production losses. Herbicide technology has been widely used by farmers as the most cost-effective weed control measure, and development of new strategy to improve herbicide tolerance in plants is urgently needed. The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to agricultural technology for crop improvement. Here we identified three polyamine uptake transporter (PUT) genes in rice that are homologous to the Arabidopsis AtRMV1. We successfully demonstrate that CRISPR/Cas9-targeted mutagenesis of OsPUT1/2/3 greatly improves paraquat resistance in rice without obvious yield penalty. Therefore, manipulation of these loci could be valuable for producing transgene-free rice with improved herbicide resistance in future.