The ATP-dependent Lon enzyme is a highly conserved protease with multiple roles in diverse species such as fungi; however, there are few reports on Lon enzymes in filamentous fungi. Thermomyces lanuginosus, a typical thermophilic fungus, has been widely studied in physiology and cell biology; thus, studies on Thermomyces Lons are important. Two Lons were bioinformatically deduced in T. lanuginosus. Subcellular localization analysis showed that one is present in mitochondria (MLon), while the other is found in peroxisomes (PLon). Although both Lon enzymes were activated by H2O2, they were not induced by heat shock; instead, they were induced by low temperatures. Two single-deletion Lon mutants (ΔMLon and ΔPLon) were generated. Biological analysis demonstrated that ΔMLon decreased the production of conidia but increased the growth of mycelia. By contrast, ΔPLon increased the production of conidia but decreased the growth of mycelia. The lifespan was measured in time and in length of continuous growth. The wild-type strain showed continuous linear growth for 60days, whereas growth was impeded at 30 and 50days for ΔPLon and ΔMLon mutants, respectively, suggesting that PLon is more important for longevity than MLon. Interestingly, ΔPLon, which accumulated larger amount of H2O2 was not only more sensitive to exogenous H2O2 but also much more sensitive to other selected stressors. Taken together, our data indicate that mitochondrial and peroxisomal Lons play opposite roles in controlling growth and development, but exhibit synergistic effects on the normal states of vegetative growth, asexual development, stress resistance and longevity in T. lanuginosus.