<p>The objective of the study was to design and characterize enzyme-responsive ufasomal nanovesicles for site-specific sustained delivery of 5-fluorouracil (5-FU) to improve therapeutic efficacy in colorectal cancer. Ethyl oleate/cholesterol ufasomes were made by thin-film hydration and optimised for particle size, zeta potential, and %EE (mean ± SD, <i>n</i> = 3). The optimised formulation (325&#xa0;mg cholesterol, 137.5&#xa0;mg ethyl oleate, 5&#xa0;mg 5-FU) was used for release studies (<i>n</i> = 3) and HCT116 cytotoxicity, apoptosis and RT-qPCR studies (<i>n</i> = 3)The optimised ufasomes had a mean particle size of 135 ± 5&#xa0;nm, a polydispersity index &lt; 0.3, a zeta potential of -32 ± 2 mV (<i>n</i> = 3), and an encapsulation efficiency of 70 ± 3.1% (<i>n</i> = 3). In vitro release studies showed minimal drug release in simulated stomach fluid (12 ± 1.2% at 2&#xa0;h), whereas β-glucosidase (5 U·mL⁻¹) triggered medication release that reached 70 ± 4.45% at 10&#xa0;h in simulated colonic fluid (pH 7.4) (<i>n</i> = 3). The free drug (<i>n</i> = 3) exhibited an IC₅₀ of 4.8 ± 0.35 µM, but the 5-FU-UF-5 showed more cytotoxicity with an IC₅₀ of 3.5 ± 0.28 µM. Treatment with 5-FU-UF-5 decreased cell migration by 88 ± 3.2%. Flow cytometry analysis revealed 58 ± 2.6% of cells in early apoptosis and 32 ± 2.1% in late apoptosis. RT-qPCR demonstrated a 5.2 ± 0.4-fold overexpression of BAX, while BCL2 and CD44 were downregulated to 0.3 ± 0.05-fold (<i>n</i> = 3). Ufasomal 5-FU administration demonstrated prolonged, targeted, and bio-responsive therapeutic efficacy in colorectal cancer, with better stability and lower systemic toxicity.</p> Graphical Abstract <p></p>