The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.