Abstract: Objective: To compare the structural characteristics and antitumor activities of pectin (PC) and its degradation products, and to analyze their structure–activity relationships. Methods: Pectin was degraded using pectate lyase, rhamnogalacturonase, and hydrogen peroxide. The structures of pectin and its degradation products were characterized by UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and high-performance liquid chromatography (HPLC). Their in vitro antitumor activities were evaluated using MTT assay, live/dead cell staining, flow cytometry, wound-healing, and cellular uptake experiments. The relationship between structural parameters and biological activity was analyzed by partial least squares (PLS) regression. Results: Three degradation products were obtained rhamnogalacturonase product (RE), pectate lyase product (PL), and hydrogen peroxide product (HPX). Compared with native pectin, all three shared the basic pectic structure but exhibited distinct molecular weight distributions, ranked as PC (530.49 kDa) > RE (381.65 kDa) > HPX (27.14 kDa) > PL (main peak 3.80 kDa). The particle size followed the order: PC (651.48 nm) > RE (612.44 nm) > PL (225.18 nm) > HPX (159.04 nm). Monosaccharide composition analysis revealed that pectin and its degradation products mainly consisted of galacturonic acid (GalA) and galactose (Gal), but with markedly different molar ratios. Comparative analysis of their antitumor activities showed that PL and HPX exhibited significantly stronger inhibitory effects on tumor cell proliferation than PC, while displaying negligible cytotoxicity toward normal cells. Both PL and HPX induced apoptosis and cell death more effectively and suppressed tumor cell migration. Cellular uptake experiments demonstrated that the degradation products exerted a synergistic effect when combined with doxorubicin (DOX), outperforming DOX alone or in combination with native pectin. PLS analysis indicated that GalA content, molecular weight, and particle size were highly correlated with antitumor activity. Conclusion: PL and HPX exhibited superior antitumor activity compared with native pectin. Their key structural features lower molecular weight, smaller particle size, and higher GalA content jointly contributed to the remarkable enhancement in bioactivity.