The side-chain oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol alter inflammatory responses to pathogenic bacteria and reduce the accessibility of plasma membrane cholesterol to prevent bacterial dissemination. However, it is not clear whether these oxysterols can also alter the intrinsic protection of cells against pore-forming toxins. Here we tested the hypothesis that 25-hydroxycholesterol and 27-hydroxycholesterol can help protect eukaryotic cells against pore-forming toxins from pathogenic bacteria. We used pyolysin, which is a cholesterol-dependent cytolysin that forms pores in multiple eukaryotic cells. Pyolysin is secreted by Trueperella pyogenes, which causes purulent infections of the bovine uterus. We found that treatment with 25-hydroxycholesterol or 27-hydroxycholesterol for 24 h protected bovine uterine cells against pyolysin-induced damage. The oxysterols reduced pyolysin-induced leakage of potassium and lactate dehydrogenase, limited cytoskeletal damage, and prevented cytolysis in both epithelial and stromal cells. The protective concentrations of 25-hydroxycholesterol and 27-hydroxycholesterol were similar to concentrations in the uterus and ovary, as determined by mass spectrometry. Treatment with 25-hydroxycholesterol or 27-hydroxycholesterol also protected human HeLa cervical cells and A549 lung cells against pyolysin-induced damage, and protected cells against Staphylococcus aureus α-hemolysin. This cytoprotection was partially dependent on the oxysterols reducing accessible cholesterol in the cell membrane. In addition, oxysterol cytoprotection was also partially inhibited after knockdown of liver X receptors with siRNA. In conclusion, 25-hydroxycholesterol or 27-hydroxycholesterol protected several types of eukaryotic cells against pore-forming toxins. Our findings imply that side-chain oxysterols could limit the severity of disease caused by pathogens that secrete pore-forming toxins.