Background The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is one of the most destructive invasive pests causing serious damage to maize crops in China. At present, the use of chemical pesticides is the main method to prevent and control S. frugiperda. However, the excessive use of chemical pesticides not only causes toxicity in humans and animals but also pollutes the environment and causes public harm. In this context, the present study aimed to isolate, identify and evaluate the pathogenicity of the native strain of Metarhizium guizhouense against eggs, larvae and pupae of S. frugiperda under laboratory conditions. Results The fungal isolate was identified as M. guizhouense (Xct1) based on its morphological and molecular characteristics by sequencing ITS, β-tubulin and EF1-α gene. Meanwhile, the fungal isolate was highly pathogenic to eggs of the S. frugiperda, at the highest concentration of 1 × 108 spores/ml, the cumulative egg hatching rate was only 8.33% at 10 days post exposure. After 10 days of exposure to the highest concentration of 1 × 108 spores/ml, the LC50 values for the first–fifth larval instars of S. frugiperda were 8.12 × 104, 1.11 × 105, 5.55 × 105, 3.87 × 106 and 1.43 × 107 spores/ml, respectively. Conclusions The pathogenicity of M. guizhouense Xct1 to S. frugiperda larvae gradually decreased with increasing the developmental stages. Metarhizium guizhouense Xct1 was more pathogenic to egg and younger larvae of S. frugiperda. This provides a basis for determining the optimal period of control.