Excessive body fat and high cholesterol are one of the leading reasons for triggering cardiovascular risk factors, obesity, and type 2 diabetes. Beta-glucan (BG)-based dietary fibers are found to be effective for lowering fat digestion in the gastrointestinal tract. However, the fat capturing mechanism of BG in aqueous medium is still elusive. In this report, we studied the dietary effect of barley-extracted BG on docosahexaenoic acid (DHA, a model fat molecule) uptake and the impact of the aqueous medium on their interactions using computational modeling and experimental parameters. The possible microscale and macroscale molecular interactions between BG and DHA in an aqueous medium were analyzed through density functional theory (DFT), Monte-Carlo (MC), and molecular dynamics (MD) simulations. DFT analysis revealed that the BG polymer extends hydrogen bonding and nonbonding interactions with DHA. Bulk simulation with multiple DHA molecules on a long-chain BG showed that a viscous colloidal system is formed upon increasing DHA loading. Experimental size and zeta potential measurements also confirmed the electrostatic interaction between BG-DHA systems. Furthermore, simulated and experimental diffusion and viscosity measurements showed excellent agreement. These simulated and experimental results revealed the mechanistic pathway of how BG fibers form colloidal systems with fat molecules, which is probably responsible for BG-induced delayed fat digestion and further halting of fatty molecule absorption in the GI tract.