This study investigates the mechanism of action of cycloheximide (CHX) and lactimidomycin (LTM), two inhibitors of eukaryotic translation elongation. CHX, a well-known protein synthesis inhibitor, has been studied for decades but its precise mechanism remains unclear. LTM, a structurally related natural product, has shown potent antiproliferative effects on tumor cell lines and selectively inhibits protein translation. A systematic comparative study revealed that both CHX and LTM block the translocation step in elongation. Footprinting experiments identified a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit as a common binding site for both inhibitors. These findings provide new insights into the molecular mechanism of inhibition of translation elongation by CHX and LTM. The study also demonstrates that LTM has antitumor activity in vivo, suggesting its potential as a novel anticancer agent.This study investigates the mechanism of action of cycloheximide (CHX) and lactimidomycin (LTM), two inhibitors of eukaryotic translation elongation. CHX, a well-known protein synthesis inhibitor, has been studied for decades but its precise mechanism remains unclear. LTM, a structurally related natural product, has shown potent antiproliferative effects on tumor cell lines and selectively inhibits protein translation. A systematic comparative study revealed that both CHX and LTM block the translocation step in elongation. Footprinting experiments identified a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit as a common binding site for both inhibitors. These findings provide new insights into the molecular mechanism of inhibition of translation elongation by CHX and LTM. The study also demonstrates that LTM has antitumor activity in vivo, suggesting its potential as a novel anticancer agent.