Chondroitin sulfate (CS) is a high molecular weight sulfated polysaccharide that has been reported to improve joint health. A recent study has demonstrated the ability of enzymatically produced CS-oligosaccharides to improve non-heme iron bioavailability. In this research, CS was chemically hydrolyzed under acid, alkaline, and combined (acid & alkaline) conditions at 60 °C for different times (0–60 h) to understand if the oligosaccharides obtained can improve iron uptake in human intestinal Caco-2 cell model. The acidic hydrolyses produced a collection of desulfated and low molecular weight CS crude oligosaccharides (CS crude oligos), while under alkaline condition a high degree of deacetylation was detected with limited depolymerisation and desulfation. The combined treated CS was first degraded and desulfated in acid solution followed by alkali deacetylation. CS crude oligos produced by the combined treatment possessed the superior antioxidant capacity measured by ferric reducing and DPPH scavenging activities. All chemically hydrolyzed CS samples showed greater (p < 0.05) ferritin formation in Caco-2 cells compared to the negative control, where the greatest ferritin level was achieved by the combined treated CS. The depolymerisation of CS followed by the deacetylation created CS structural changes resulting in the increase of antioxidant and Fe-absorption enhancing capacities.