Neomycin bind specifically to the 30S ribosomal subunit and inhibit translation.

Neomycin is an aminoglycosid antibiotic. As such, it acts primarily by interfering protein synthesis due to high affinity binding to the 30s portion of bacterial ribosomes. This causes not only a blockade in protein synthesis, but also translation errors (due to mRNA missreading), leading to aberrant, non-functional proteins. In order to bind to their target, aminoglycosides have to first penetrate inside the bacterial cells, in a process that consumes energy provided primarily by electron transfer through the aerobic respiratory chain, being this the main reason why these antibiotics are not active against anaerobic bacteria. Once the antibiotic begins to interfere with protein synthesis and translation, the bacterial cell wall starts to lose integrity, allowing for a massive, energy-independent entry of aminoglycosides, which ultimately results in bacterial cell lysis (bactericidal effect). Bacterial resistance to aminoglycosides can arise mainly from i) mutations in the ribosomal targets of these antibiotics, ii) acquisition of different types of enzymes that inactivate aminoglycosides (via adenylation, phosphorylation or acetylation), iii) modifications in the bacterial cell wal leading to impermeability against these antibiotics. Less frequently, bacteria can also resist aminoglycisdes by acquiring efflux pumps ablel to extrude the antibiotic molecules to the outside.