The anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract was examined. Uptake of rosemary extract fractions was tested on Caco-2 cell monolayers (2–12 h incubation times) and the quantification of carnosic acid and carnosol was performed by UPLC-MS/MS. Human macrophages were treated with the basolateral fractions and secretion of TNF-α, IL-1β, IL-6 and IL-10 was measured by ELISA. The fractions obtained after 8 and 12 h in absorption experiments caused a significant reduction in excretion of the pro-inflammatory cytokines. This reduction in cytokine secretion levels corresponded to the amounts of carnosic acid and carnosol in the basolateral fractions. Thus, the basolateral fraction of a rosemary supercritical extract showed an important anti-inflammatory activity, providing the basis for increasing the use of supercritical rosemary extracts for the prevention of inflammatory diseases.