USP28-IN-3 is a USP28 inhibitor ( IC 50 =0.1 μM) with high selectivity over USP2 , USP7 , USP8 , USP9x, UCHL3 and UCHL5. USP28-IN-3 shows cytotoxicity against cancer cells, down-regulates the cellular level of c-Myc through ubiquitin-proteasome system. USP28-IN-3 also decreases the ankyrase-1/2 level in vitro. USP28-IN-3 enhance the sensitivity of colorectal cancer cells to Regorafenib.