Targeted contrast agents (CAs) can improve magnetic resonance imaging (MRI) for accurate cancer diagnosis. In this work, we used the Shiga toxin B-subunit (STxB) as a targeting agent, which binds to Gb3, a glycosphingolipid highly overexpressed on the surface of tumor cells. We developed STxB-targeted MRI probes from cyclic peptide scaffolds functionalized with six to nine monoamide DO3A[Gd(III)] chelates. The influence of structural constraints on the longitudinal relaxivity (r1) of the CAs has been studied. The cyclic peptide carrying nine monoamide DO3A[Gd(III)] exhibited a r1 per compound of 32 and 93 mM-1s-1 at 9.4 and 1.5 T, respectively. Its conjugation to the pentameric STxB protein led to a 70 kDa compound with a higher r1 of 150 and 475 mM-1 s-1 at 9.4 and 1.5 T, respectively. Specific accumulation and cellular distribution of this conjugate in Gb3-expressing cancer cells were demonstrated using immunofluorescence microscopy and quantified by an inductively coupled plasma-mass spectrometry dosage of Gd(III). Such an agent should enable the in vivo detection by MRI of tumors expressing Gb3 receptors.