Colorectal cancer ranks second in cancer-related deaths globally. 5-fluorouracil (5-FU) is a common first-line chemotherapy drug, but acquired or intrinsic resistance to the drug remains a significant challenge. Gelsolin, an actin-binding cytoskeletal protein, is known to be involved in anti-apoptotic pathways and overexpression in cancer cells promotes their migration and invasion. Our work investigates the role of gelsolin in chemoresistance to 5-FU in colorectal cancer. Employing gain and loss of function models, we examined the role of gelsolin in 5-FU resistance via clonogenic assays and Western blot analysis, on gelsolin overexpression (OE), empty vector (EV), wild type (WT) and knock-out (KO) HCT116 colorectal cancer cells. Increased colony size and number were observed with higher gelsolin expression in clonogenic assays, showing less cell death and more colonies formed under treatment with 5-FU. Additionally, cells with elevated gelsolin levels showed reduced indicators of apoptosis, such as cleaved caspase-3 and PARP-1 in Western blot analysis. We conclude that lower gelsolin expression increases 5-FU-induced apoptosis in colorectal cancer. Our findings highlight gelsolin's role in 5-FU resistance in colorectal cancer, indicating its potential as a supplementary chemotherapy target and prognostic marker for 5-FU treatment. We also propose gelsolin as an adjunctive drug target and prognostic marker for colorectal cancer with 5-FU treatment.