The lipid-lowering activity of fucoidan has been widely reported, but the exploration of its mechanisms is relatively limited, and studies on its direct targets are even scarcer. Additionally, it is unclear whether different administration methods affect the lipid-lowering activity of fucoidan. In current study, we used fucoidan derived from Saccharina japonica (SJF) to investigate its targets. The results showed that not only did SJF directly inhibit the Niemann-Pick C1-like 1 (NPC1L1)-mediated cholesterol transport, but it also reduced the solubility of cholesterol in mixed micelles, thereby interfering with the cholesterol uptake. Furthermore, SJF not only directly inhibited the activity of pancreatic triglyceride lipase (PTL), but also interfered with the overall catalytic process facilitated by colipase, thereby reducing the absorption of triglycerides. Moreover, comparative studies on the lipid-lowering activity of SJF administered via different methods demonstrated that dietary supplementation with SJF provided better lipid-lowering effects compared with gavage administration. Our research not only elucidates the targets and mechanisms of SJF but also provides theoretical basis for the selection of administration methods for fucoidan in lipid-lowering therapy.