In this study, a novel series of 4,6,7-trisubstituted quinoline analogues bearing thiazolidinones were designed and synthesized based on our previous study. Among them, the most potent compound 15i, 4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)-N,N-diethylpiperidine-1-carboxamide was identified as a multi-kinase inhibitor. The results of MTT assay revealed in vitro antitumor activities against HT-29 cells of compound 15i with an IC value of 0.19 μM which was 14.5-fold more potent than that of Regorafenib. In the cellular context, significant antiproliferation, cytotoxicity and induction of apoptosis on HT-29 cells in a dose- and time-dependent manner were confirmed by IncuCyte live-cell imaging assays. Moreover, compound 15i strongly induced apoptosis by arresting cell cycle into the G2/M phase. No antiproliferation and cytotoxicity against human normal colorectal mucosa epithelial cell FHC was observed at 10.0 μg/mL or lower concentrations which indicated that the toxicity to normal cells of compound 15i was much lower than that of Regorafenib. Based on the above findings, further structural modification will be conducted for the development of more potent kinase inhibitors as anticancer agents.