Mixed populations of domesticated and feral pig gut microorganisms (Recombined Porcine-Derived Continuous Flow culture; RPCF and Feral Culture; FC, respectively) were grown in continuous culture to investigate the effects of tylosin on antimicrobial resistance. Cultures established in steady state were inoculated with 9.7 log10 colony forming units (CFU) of a tylosin-resistant Enterococcus faecium and allowed 7 days to re-establish equilibrium before administration of 100 µg tylosin mL-1. Total culturable anaerobes recovered on non-antibiotic supplemented medium, thus inclusive of tylosin-sensitive and -insensitive bacteria, ranged from 7.15 to 9.20 log10 CFU mL-1 throughout 8 days of tylosin administration and 6 subsequent days without tylosin administration. Recovery of total anaerobes on tylosin-supplemented medium revealed that populations of total tylosin-insensitive anaerobes ranged from 6.30 to 9.02 log10 CFU mL-1 during the experiment. Concentrations of the introduced tylosin-resistant E. faecium decreased to near minimum detectable levels (1.3 log10 CFU mL-1) in the cultures before initiation of tylosin administration and then increased to 6.80 ± 0.28 and 8.30 ± 0.43 log10 CFU mL-1 in RPCF and FC cultures, respectively, and remained higher than day 0 concentrations for the remainder of the experiment. Endogenous tylosin-insensitive Enterococcus were undetectable before administration of tylosin but tylosin-resistant E. faecalis and E. hirea found to have acquired an ermB gene of expected size and sequence of that contained in the introduced E. faecium were enriched to 7.74 ± 0.37 and 3.85 ± 4.03 after initiation of tylosin administration. These results demonstrate the acquisition, propagation and persistence of tylosin-resistance in mixed populations of domestic and feral swine gut microflora.