Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.