The entomopathogenic fungus Metarhizium anisopliae produces several chitinases. Due to the diversity of these enzymes, investigations to assign a specific function to each enzyme are still on-going. It is well documented that a chitinase (CHI2) play a critical role in early stages of infection, especially in invading of insect’s cuticle and penetration. In this study, the expression of CHI2 gene was studied during the different stages of infection of fungus M. anisopliae when infecting Spodoptera litura larvae and after transfer to artificial culture medium using quantitative real-time RT-PCR. Larvae samples were collected at different stages: i) from un-infected larvae (0 h); ii) from infected larvae at 2, 12, and 24 hours post-infection and when larvae were in moribund stage (Moribund larvae were collected at 60 hours after infection in S. litura); iii) from cadavers when only mycelia were detected on cadavers, when few conidia were observed on mycelia, and when cadavers were covered with conidia; iv) from artificial culture media (dry conidia were produced on rice as a substrate and mycelia were obtained from liquid culture). Transcript levels of the CHI2 gene was first normalized to three housekeeping genes. Normalized data were then calibrated to the transcript level of sample 12 h. Transcripts were not detected in the control uninfected larvae and 2 h sample. CHI2 was detected after 12 hours, but its expression increased significantly after moribund stage. High expressions of CHI2 gene were observed when fungal mycelia emerged and conidiation started on the insect cadaver. Expression level decreased markedly when the fungus was cultured in the culture media. In the present study, the high expression of this gene during the late stages of pathogenesis when mycelia emerged and subsequently conidiated on the cadavers obviously shows their important roles at the post-mortem stages too. It is hypothesized that as host nutrient is consumed, these enzymes are up-regulated to facilitate invading of cuticle and emerging of hypha from the cuticle. Moreover, this enzyme may involve in providing the essential nutrients for mycelium growth and conidiation.