The cytoplasmic domain of the membrane-anchored human EGF proform is encoded by exons 2224 and the function of this proEGFcyt is largely unkown. Stable transfectants of the human thyroid carcinoma (Ca) cell line FTC-133 were generated over-expressing (a) proEGFcyt, (b) truncated peptide version encoded by exons 22 and 23 (proEGF22.23), and (c) a natural splice version with a deletion of exon 23 and 24 (proEGFdel23). ProEGFcyt and proEGF22.23 transfectants, but not proEGFdel23 or mock clones, demonstrated a significant reduction in growth rates. This coincided with a marked post-translational reduction in EGFR and ErbB2 protein levels in proEGFcyt and proEGF22.23 clones. Incubation of these transfectants with the proteasome inhibitors MG132 and lactacystin diminished the growth inhibitory effect of proEGFcyt and reversed the down-regulation of EGF receptor proteins suggesting an involvement of the ubiquitin-proteasome system. Microarray analysis of FTC-133-proEGFcyt clones revealed a strong downregulation of the ubiquitin C-terminal hydrolase-L1 (UCH-L1), thus, identifying proEGFcyt as a new regulator of this de-ubiqitinating enzyme. Silencing of UCH-L1 gene activity and the absence of UCH-L1 protein was exclusively observed in proEGFcyt and proEGF22.23 FTC-133 transfectants but absent in proEGFdel23 and mock controls implicating a possible involvement of exon23 encoded peptide of proEGFcyt. Westernblot analysis of total ubiquitinated protein revealed significantly increased levels of cellular ubiquitinated proteins in proEGFcyt and proEGF22.23 transfectants. Specific knockdown of UCH-L1-containing proEGFdel23 and mock clones resulted in reduced EGFR protein levels similar to proEGFcyt and this decrease in EGFR was prevented in the presence of MG132. In summary, we present evidence for a novel and unique mechanism for the growth inhibitory actions of proEGFcyt in human thyroid Ca cells. We identified proEGFcyt as a new regulator of the de-ubiquitinating enzyme UCH-L1 and demonstrate that proEGFcyt-mediated silencing of UCH-L1 causes the decrerase in EGFR likely as a result in post-translational hyperubiquitination of EGFR in human thyroid Ca cells. These findings may have important implications for the design of new treatments particularly of undifferentiated thyroid cancer.