Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed alpha-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.