Background and Purpose Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes. However, there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria. Experimental Approach Different bacterial strains were incubated with Td, and the bacterial uptake efficiency of Td was measured by flow cytometry and confocal microscopy. Then we designed two types of linkages modes between Td and ASOs targeting gfp, encoding green fluorescent protein , or acpP, encoding the acyl carrier protein, and assessed the efficiency of delivery by flow cytometry and gene knockdown by RT-PCR, colony counting and confocal microscopy in E. coli. Key results Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Lipofectamine 2000 (LP2000) in a 0.125 μL/μg ratio to Td helped Td penetrate into bacteria, but had no apparent effect on linear DNA in the same concentration. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure. Conclusion and Implications This study established an effective antisense delivery system based on loop-armed Td, which open opportunities for developing antisense antibiotics.