Hypoxia plays a significant role in tumor angiogenesis and proliferation. Hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia is overexpressed in a number of malignant tumor types. RNA interference (RNAi) is effective to silence the expression of cancer related genes for cancer therapy. We have developed a novel pH-sensitive multifunctional lipid delivery system (ECO) for systemic targeted delivery of small interfering RNA (siRNA) to silence target proteins for cancer therapy. In this study, we assessed the effectiveness of RGD peptide targeted ECO/siRNA nanoparticles against HIF-1α in mouse HT29 colon cancer xenografts with DCE-MRI and a biodegradable macromolecular contrast agent. A mouse model bearing subcutaneous HT29 colon adenocarcinoma flank xenografts was developed by inoculation of 5x105 cells into athymic nude mice. Before the start of treatment, tumors were allowed to grow for two weeks, until they were approximately 0.5 cm in diameter. The mice then were intravenously injected with RGD-targeted ECO/siHIF-1α nanoparticles at a dose of 2.0 mg/kg every 3 days. Another group received PBS as a control. DCE-MRI was performed on each mouse from both the saline control and RGD-targeted ECO/siHIF-1α treatment groups. Each mouse was catheterized and injected with a bolus of GODP, a polydisulfide biodegradable macromolecular contrast agent containing Gd-DOTA monoamide. A T1-weighted 3D-FLASH gradient echo sequence was used for the DCE-MRI data acquisition on a 7T pre-clinical scanner (Bruker) on day 21 after the start of the therapy. DCE-MRI data were analyzed by fitting with the tissue homogeneity (AATH) model the adiabiatic approximation.The treatment with RGD targeted ECO/siHIF-1α nanoparticles resulted in significant silencing of HIF-1α and inhibition of tumor growth as compared to the control group. Silencing of HIF-1α also resulted in significant decreases in VEGF and CD31 expression in tumor, indicating the anti-angiogenic capabilities. DCE-MRI revealed significant reduction in tumor vascularity when treated with the targeted ECO/siHIF-1α nanoparticles. There was over 70% reduction in average tumor blood flow (Fp), permeability-surface area product (PS), and plasma volume fraction (Vp) in the treatment group as compared to the saline control group (p < 0.05). Histological analysis showed substantially more necrotic tissue in the core of the ECO/siHIF-1α treated tumors than in those of the control group, validated the DCE-MRI data.The targeted ECO/siHIF-1α nanoparticles induced a multi-faceted knockdown of HIF-1α, VEGF, and CD31, resulting in tumor growth inhibition. DCE-MRI with GODP revealed that silencing of HIF-1α expression was effective to inhibit tumor angiogenesis in cancer therapy.