Since its first description in 1966, macrophage migration inhibitory factor (MIF) was found to play a critical role in inflammatory and immune responses as well as in disease pathogenesis especially in tumor pathogenesis and cancer progression. MIF is expressed in different cell types and is associated with many disease severity and tumor pathogenesis. Here, we investigated the influence of TLR7 and TLR8 agonist resiquimod (R848), an immune response inducer used as a prophylactic agent for several infectious diseases as well as anticancer agents and vaccine adjuvant on MIF expression in cells and organs. Humans, mice and rats cell lines from different tissues (blood, retinal, nasopharynx, brain and liver) and C57BL/6J mice organs (brain, liver and spleen) were used for this investigation. In vitro, R848 induced MIF gene overexpression except in brain and liver cells. Furthermore, it enhanced cells ability to release soluble MIF and differently regulated mRNA expression of MIF-related receptors (CD74, CXCR4, CXCR2 and CD44). Its influence on MIF gene expression and MIF proteins release was more consistent in cancer cells. In vivo, a strong positive expression of MIF was observed in different regions in brain and spleen in response to R848 treatment; however in liver, increased MIF expression was observed in hepatocytes only. On the other hand, R848 treatment had induced a slight enhancement of MIF concentration in the plasma of C57BL/6J mice. Taken together, these data suggest that R848 differently regulates MIF mRNA expression depending on organ types and could influence MIF concentration in cellular microenvironment.