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<i>Purpureocillium lilacinum</i> has been widely used as a commercial biocontrol agent for the control of plant parasitic nematodes. Whole genome analysis promotes the identification of functional genes and the exploration of their molecular mechanisms. The <i>Cyclophilin</i> (<i>CYP</i>) gene family belongs to the immunophillin superfamily, and has a conserved cyclophilin-like domain (CLD). CYPs are widely identified in prokaryotes and eukaryotes, and can be divided into single- and multi-domain proteins. In the present study, 10 <i>CYP</i> genes possessing the CLD, named <i>PlCYP1&#8722;P10</i>, were identified from the genome of <i>P. lilacinum</i> strain 36-1. Those 10 PlCYPs were predicted to have different cellular localizations in <i>P. lilacinum</i>. Phylogenetic and gene structure analysis revealed the evolutionary differentiation of CYPs between Ascomycotina and Saccharomycotina fungi, but conservation within the Ascomycotina fungi. Motif and gene structure distributions further support the result of phylogenetic analysis. Each <i>PlCYP</i> gene had a specific expression pattern in different development stages of <i>P. lilacinum</i> and its parasitism stage on eggs of <i>Meloidogyne incognita</i>. In addition, the 10 <i>PlCYP</i> genes exhibited different expression abundances in response to abiotic stresses, among which <i>PlCYP4</i> was highly expressed at a high temperature (35 &#176;C), while <i>PlCYP6</i> was up-regulated under 5 mM of H₂O₂ stress. Furthermore, the heterologous expression of <i>PlCYP4</i> and <i>PlCYP6</i> in <i>Escherichia coli</i> enhanced the cellular tolerance against a high temperature and H₂O₂. In summary, our study indicates the potential functions of <i>PlCYPs</i> in virulence and the stress response, and also provides a frame for further analysis of the <i>CYP</i> gene family in Ascomycotina fungi.