, a specialized fungal pathogen, causes lethal infection in honeybee larvae. miRNA-like small RNAs (milRNAs) are fungal small non-coding RNAs similar to miRNAs, which have been shown to regulate fungal hyphal growth, spore formation, and pathogenesis. Based on the transcriptome data, differentially expressed miRNA-like RNAs (DEmilRNAs) in infecting the worker 4-, 5-, and 6-day-old larvae (Aa-4, Aa-5, and Aa-6) were screened and subjected to trend analysis, followed by target prediction and annotation as well as investigation of regulatory networks, with a focus on sub-networks relative to MAPK signaling pathway, glycerolipid metabolism, superoxide dismutase, and enzymes related to chitin synthesis and degradation. A total of 606 milRNAs, with a length distribution ranging from 18 nt to 25 nt, were identified. The first nucleotide of these milRNAs presented a bias toward U, and the bias patterns across bases of milRNAs were similar in the aforementioned three groups. There were 253 milRNAs, of which 68 up-and 54 down-regulated milRNAs shared by these groups. Additionally, the expression and sequences of three milRNAs were validated by stem-loop RT-PCR and Sanger sequencing. Trend analysis indicated that 79 DEmilRNAs were classified into three significant profiles (Profile4, Profile6, and Profile7). Target mRNAs of DEmilRNAs in these three significant profiles were engaged in 42 GO terms such as localization, antioxidant activity, and nucleoid. These targets were also involved in 120 KEGG pathways including lysine biosynthesis, pyruvate metabolism, and biosynthesis of antibiotics. Further investigation suggested that DEmilRNA-targeted mRNAs were associated with the MAPK signaling pathway, glycerolipid metabolism, superoxide dismutase, and enzymes related to chitin synthesis and degradation. Moreover, the binding relationships between aap-milR10516-x and as well as between aap-milR-2478-y and 1 were confirmed utilizing a combination of dual-luciferase reporter gene assay and RT-qPCR. Our data not only provide new insights into the proliferation and invasion, but also lay a basis for illustrating the DEmilRNA-modulated mechanisms underlying the infection.