Ractopamine HCl (RHC) is supplemented to feedlot cattle to improve feed efficiency and increase carcass weight. Supplementation of RHC clearly benefits livestock production, but it is of note that the adrenergic system through which it acts is typically associated with stress. The purpose of this study was to identify changes in the transcriptome of whole blood in RHC-supplemented feedlot cattle. We hypothesized that transcripts related to inflammatory processes would be upregulated after 35 days of dietary RHC supplementation. To test this hypothesis, RNA from whole blood collected from 16 cattle before and after supplementation with 300 mg/day of RHC was sequenced using 3' tag-seq. Eight transcripts were differentially expressed (Adjp < 0.10) between pre- and post-supplementation blood samples. Although several of these transcripts including IFI35, TYROBP, and TP53INP1 are associated with inflammation, a systemic dysregulation of inflammatory pathways was not evident. These data provide insight into the response of cattle to RHC supplementation that will direct future studies examining how the transcriptome of whole blood and other tissues responds during acute exposure to RHC and how this supplement mechanistically improves growth performance.