Two series of novel urea-containing peptide boronic acids were designed and synthesized as proteasome inhibitors. The SAR was investigated and one of the two series showed impressive anti-proteasome and anti-tumor activities. More human tumor cell lines were tested and compound I-14 was selected to evaluate its anticancer activity in vivo. Compound I-14 showed obvious in vivo anticancer activity with much lower toxicity comparing to Bortezomib.