Equine influenza is a common disease of the horse, causing significant morbidity worldwide. Here we describe the establishment of a plasmid-based reverse genetics system for equine influenza virus. Utilizing this system, we generated three mutant viruses encoding carboxy-terminally truncated NS1 proteins. We have previously shown that a recombinant human influenza virus lacking the NS1 gene (delNS1) could only replicate in interferon (IFN)-incompetent systems, suggesting that the NS1 protein is responsible for IFN antagonist activity. Contrary to previous findings with human influenza virus, we found that in the case of equine influenza virus, the length of the NS1 protein did not correlate with the level of attenuation of that virus. With equine influenza virus, the mutant virus with the shortest NS1 protein turned out to be the least attenuated. We speculate that the basis for attenuation of the equine NS1 mutant viruses generated is related to their level of NS1 protein expression. Our findings show that the recombinant mutant viruses are impaired in their ability to inhibit IFN production in vitro and they do not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in MDCK cells, or in vivo in a mouse model. Therefore, these attenuated mutant NS1 viruses may have potential as candidates for a live equine influenza vaccine.