The cellular tropisms of eukaryotic viruses are shaped by their need for entry receptors and intracellular transcription factors. Here we show that viral tropisms can also be regulated by tissue-specific microRNAs (miRNAs). Target sequences complementary to muscle-specific miRNAs were inserted into the 3' untranslated region (UTR) of an oncolytic picornavirus that causes lethal myositis in tumor-bearing mice. The recombinant virus still propagated in subcutaneous tumors, causing total regression and sustained viremia, but could not replicate in cells expressing complementary miRNAs and therefore did not cause myositis. This altered tropism was not due to insertional attenuation, as a control virus containing a 3' UTR insert with a disrupted miRNA target sequence fully retained its lethal myotropism. Tissue-specific destabilization of viral genomes by miRNA target insertion provides a potentially versatile new mechanism for controlling the tropism of replicating viruses for therapy and may serve as a new modality for attenuating viruses for vaccine purposes.