Picornavirus’ genomic RNA is a positive-stranded RNA sequence that also serves as a template for translation and replication. Cellular microRNAs were reported to interfere to different extents with the replication of specific picornaviruses, mostly acting as inhibitors. However, owing to the high error rate of their RNA-dependent RNA-polymerases, picornavirus quasi-species are expected to evolve rapidly in order to lose any detrimental microRNA target sequence. We examined the genome of Theiler’s murine encephalomyelitis virus (TMEV) for the presence of under-represented microRNA target sequences that could have been selected against during virus evolution. However, little evidence for such sequences was found in the genome of TMEV and introduction of the most under-represented microRNA target (miR-770-3p) in TMEV did not significantly affect viral replication in cells expressing this microRNA. To test the global impact of cellular microRNAs on viral replication, we designed a strategy based on short-term Dicer inactivation in mouse embryonic fibroblasts. Short-term Dicer inactivation led to a >10-fold decrease in microRNA abundance and strongly increased replication of Vesicular stomatitis virus (VSV), which was used as a microRNA-sensitive control virus. In contrast, Dicer inactivation did not increase TMEV replication. In conclusion, cellular microRNAs appear to exert little influence on Theiler’s virus fitness.