Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.