Fosfomycin (FOS) is an antibiotic used, mostly in Latin America, for the treatment of lung and enteric infections of pigs. Intracellular fluids of enterocytes can act as biophase for Lawsonia intracellularis, the causative agent of porcine proliferative enteropathy (PPE). The aim of this study was to determine whether the presence of L. intracellularis in the enterocytes modifies FOS penetration. Eight healthy pigs in growth-finishing stage were used to produce healthy (group A) and L. intracellularis-colonized (group B) intestinal explants. For both groups, treatment consisted of a 580 μg/ml concentration of calcium FOS, which was added to each explant (0.5-6 hr). For group B, the Enterisol Ileitis vaccine was used as source of the micro-organism. Previously to the assay, the time necessary for L. intracellularis to colonize the enterocytes was defined. Also, a PCR protocol was optimized to determine the presence of the pathogen in the explants. There were nonstatistical differences for the penetration of the antibiotic into healthy and L. intracellularis-colonized enterocytes. MIC of FOS for L. intracellularis is unknown; nevertheless, MIC of various antibiotics ranges between 0.125 and 128 μg/ml. FOS reaches inside the enterocyte concentrations which surpass the MICs of other antibiotics that also act by the inhibition of cell wall synthesis; however, further studies should be carried out to determine fosfomycin MIC for L. intracellularis to discern the usefulness of this antibiotic in the treatment of PPE.