In the current study, we explored for the first time, the mechanism of action of the new Casein kinase 1 $\varepsilon$ (CK1$\varepsilon$) selective inhibitor GSD0054. Although GSD0054 behaved as a selective CK1$\varepsilon $ inhibitor in enzymatic assays, we studied whether this inhibitory activity also occurred inside the cells. The effects of GSD0054 on $\beta $-catenin expression and disruption of cell cycle progression were studied in the human breast cancer cell lines MDA-MB-453 ($\beta $-catenin negative) and T-47D ($\beta $-catenin positive). We also performed molecular modeling studies using computational docking against CK1$\varepsilon $ to explain and predict the mechanism of action of this compound. Moreover, the commercially available CK1$\varepsilon $ inhibitor PF-4800567 and the CK1$\delta $/$\varepsilon $ inhibitors PF-670462 and IC261 were also studied for comparison purposes.GSD0054 showed anti-proliferative activity against MDA-MB-453 and T-47D cells despite the fact that MDA-MB-453 cells do not possess active $\beta$-catenin. However, selective cell killing occurred in the more resistant, $\beta $-catenin active, T-47D cells. CK1$\varepsilon $ was confirmed as a cellular target, although other targets or alternative mechanisms of action could possibly explain the anti-proliferative activity in MDA-MB-453 cells.