AbstractKeep that motor running: Type IV pili are among the strongest molecular motors characterized to date. Herein it is reported that pilus motors of the human pathogen Neisseria gonorrhoeae are very active for at least one day post‐infection of epithelial cells. They generate force in the range on 70 pN and retract at a higher velocity as compared to abiotic environments (see picture).magnified imageType IV pili are important bacterial virulence factors that mediate attachment to mammalian host cells and elicit downstream signals. When adhered to abiotic surfaces, the human pathogen Neisseria gonorrhoeae generates force by retracting these polymeric cell appendages. We recently found that single pili generate stalling forces that exceed 100 pN, but it is unclear whether bacteria generate force once they adhere to their human host cells. Here, we report that pili retract very actively during infection of human epithelial cells. The retraction velocity is bimodal and the high velocity mode persisted at higher forces in contrast to an abiotic environment. Bacteria generate considerable force during infection, but the maximum force is reduced from 120±40 pN on abiotic surfaces to 70±20 pN on epithelial cells, most likely due to elastic effects. Velocity and maximum force of pilus retraction are largely independent of the infection period within 1 h and 24 h post‐infection. Thus, the force generated by type IV pili during infection is high enough to induce cytoskeletal rearrangements in the host cell.