Cycloheximide (CHX) and lactimidomycin (LTM) are inhibitors of eukaryotic translation elongation. CHX, a well-known protein synthesis inhibitor, blocks the translocation step in elongation by binding to the ribosome and inhibiting eEF2-mediated translocation. LTM, a related compound, also blocks translocation but acts through a similar yet distinct mechanism. Both compounds protect a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, defining a common binding pocket. These findings suggest that both CHX and LTM inhibit translation elongation by binding to the same site on the ribosome, though with different affinities. LTM is more potent than CHX, inhibiting protein synthesis more effectively. LTM also exhibits antitumor activity in vivo, indicating its potential as a novel anticancer agent. The study reveals that LTM and CHX inhibit eukaryotic translation elongation by blocking the translocation step, with LTM acting at an earlier stage than CHX. The results highlight the importance of the 12-membered macrocycle in the mechanism of action of these inhibitors. The common binding site on the 60S ribosome for both CHX and LTM, along with the cross-resistance of yeast strains to both compounds, suggests that they may share a similar mechanism of action. The study also demonstrates that LTM inhibits breast cancer growth in vitro and in vivo, supporting its potential as a therapeutic agent. The findings provide a comprehensive understanding of the molecular mechanisms of action of CHX and LTM in inhibiting eukaryotic translation elongation.Cycloheximide (CHX) and lactimidomycin (LTM) are inhibitors of eukaryotic translation elongation. CHX, a well-known protein synthesis inhibitor, blocks the translocation step in elongation by binding to the ribosome and inhibiting eEF2-mediated translocation. LTM, a related compound, also blocks translocation but acts through a similar yet distinct mechanism. Both compounds protect a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, defining a common binding pocket. These findings suggest that both CHX and LTM inhibit translation elongation by binding to the same site on the ribosome, though with different affinities. LTM is more potent than CHX, inhibiting protein synthesis more effectively. LTM also exhibits antitumor activity in vivo, indicating its potential as a novel anticancer agent. The study reveals that LTM and CHX inhibit eukaryotic translation elongation by blocking the translocation step, with LTM acting at an earlier stage than CHX. The results highlight the importance of the 12-membered macrocycle in the mechanism of action of these inhibitors. The common binding site on the 60S ribosome for both CHX and LTM, along with the cross-resistance of yeast strains to both compounds, suggests that they may share a similar mechanism of action. The study also demonstrates that LTM inhibits breast cancer growth in vitro and in vivo, supporting its potential as a therapeutic agent. The findings provide a comprehensive understanding of the molecular mechanisms of action of CHX and LTM in inhibiting eukaryotic translation elongation.