Depolymerized Fucoidan derivatives (DFd) are emerging anticancer agents, yet their relationship with heparanase (HPSE) remains unclear. H ₂ O ₂ -assisted depolymerization of native fucoidan ( Fucus vesiculosus ) generates DFd characterized by reduced number- and weight-average molecular masses (M _n and M _w ) and degree of sulfation (DS) with oxidative structural modifications, resulting in heterogeneous yet bioactive compounds with reduced anticoagulant activity compared to the native fucoidan. Their inhibitory effect on HPSE in vitro was associated with both M _n and DS, although no dominant factor was identified. Biologically, DFd decreased mitochondrial metabolic activity and migration in breast cancer models according to M _n and DS reductions, with differential responses between MCF7 and MDA-MB-231 cells. Despite minor cell line-specific changes in HPSE expression, heparan sulfate (HS) levels remained unchanged, suggesting that these effects are not driven by altered HPSE expression or extracellular matrix degradation. This study provides a key original contribution by tracking DFd cellular internalization for the first time in these cell lines, revealing a dependence on M _n /DS and a correlation with the observed migration inhibition for the MCF7 cells treated with the smallest DFd. These findings highlight cellular uptake as a critical determinant of fucoidan bioactivity and open new perspectives for understanding and optimizing their anticancer mechanisms.