Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in Delta Bbecm33 (76 %) than in Delta Mrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in Delta Bbecm33 only. Consequently, Delta Bbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than Delta Mrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca2+ during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in Delta Bbecm33 but was unaffected in Delta Mrecm33, unlike a similar decrease (25-28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither Delta Bbecm33 nor Delta Mrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.