In order to study the effects of miRNAs in stripped snakehead (SSN-1) cell line upon snakehead vesiculovirus (SHVV) infection, SHVV-infected and mock-infected SSN-1 cells were analyzed by miRNA high-throughput sequencing. The expression levels of twenty-two miRNAs with levels equal to or higher than 1 000 reads per million (TPM ≥ 1 000) were determined by qRT-PCR with U6, β-actin, and 5S rRNA as internal control. When U6 gene was used as internal control, no significant changes were observed for β-actin upon SHVV infection, while 5S rRNA was upregulated in SSN-1 cells upon SHVV infection. The results showed that U6 gene or β-actin could be used as internal control genes for miRNAs in SSN-1 cells. Furthermore, fourteen miRNAs were selected and their effects on SHVV replication were analyzed. The results showed that eleven miRNAs (miR-27a-3p, miR-26a-5p, miR-30e-3p, etc.) promoted, whereas miR-150 and miR-216b suppressed, SHVV replication. Next, we found that high-expression miR-100-5p was upregulated at early stage of SHVV infection, while downregulated at later stage. Overexpression of miR-100-5p exhibited significant suppression of SHVV replication, while inhibition of expression of miR-100-5p promoted SHVV replication. Our findings presented valuable information for the study of molecular drugs against SHVV.