Next-generation RNA constructs are emerging as exciting new platforms for both vaccination and therapeutic applications. The last few years have seen the first mRNA vaccines licensed for use in humans and the first-in-human clinical trial of a self-amplifying RNA vaccine. With their short time-to-market, rapid manufacture, low cost, and ultra-low dosage, saRNA vaccines are potentially a game-changer when it comes to dealing with novel disease outbreaks. Whilst the pre-clinical testing of a VEEV-derived saRNA vaccine yielded robust neutralisation and 100% seroconversion, the results from the first-in-human trial were notably more muted, with poor neutralisation and failure to induce 100% seroconversion. Our aim, therefore, was to investigate why humans and mice respond so differently to the VEEV saRNA construct; we hypothesised that there is a difference in the innate immune responses to saRNA in the cells that are responsible for taking up saRNA and synthesising antigen at the site of injection, i.e. muscle tissue. To investigate this, we used a transcriptomics approach to characterise the response of human muscle cells to VEEV saRNA and compared it with the transcriptomic changes seen in murine muscle cells. We observed that both human and mouse muscle cells produce a robust innate immune response to VEEV saRNA; however, there were several notable differences. Murine cells elevated the expression of cytokines and chemokines to a far greater extent than human muscle cells, while the expression of IRF7, a key transcription factor for orchestrating the antiviral response, was markedly higher in humans. Overall, these results indicate that there are distinct differences in the way that human cells react to saRNA compared to murine cells and that this should be taken into account when designing and testing saRNA vaccines in the future.