Rationale and Objectives: The aim of this study was to assess the feasibility of thermally cross-linked superparamagnetic iron oxide nanoparticle contrast (TCL-SPION) in magnetic resonance (MR) imaging (MRI) for the detection of lymph node metastasis in experimental model. Materials and Methods: B16F1 human melanoma cells were subcutaneously injected into the thighs of C57B116 mice (n = 10). MRI was performed 21 days after tumor injection using a 4.7-T MR scanner. In vivo MRI was performed before and after the intravenous administration of TCL-SPION using T2 fast spin-echo and T2 gradient-echo pulse sequences. Then, ex vivo MR images were obtained for resected inguinal lymph nodes (n = 18) using the same pulse sequences as for in vivo imaging. On the basis of hematoxylin and eosin staining results, the lymph nodes were classified into three groups: group 1, nonmetastatic; group 2, tumor volume < 50% of the resected sample; and group 3, tumor volume > 50% of the resected sample. Size, signal-to-background ratio, and enhancement pattern were evaluated in each of the three groups on ex vivo images. Results: The findings observed on ex vivo MR images of 18 inguinal lymph nodes were compared with histopathologic findings. All nodes were classified into three groups: group 1, n = 6; group 2, n = 5; and group 3, n = 7. The sizes of the lymph nodes in group 1 were significantly different from the sizes of those in group 3 (P = .014), but there was no significant difference in lymph node sizes between groups 1 and 2 (P = .792). Signal-to-background ratios of samples in groups 2 and 3 were significantly higher than those of samples in group 1 (P = .045 and P = .007, respectively). Each group of lymph nodes showed characteristic enhancement patterns that were well correlated between the images and pathology, except for one node. Conclusions: The features and extent of metastasis in the lymph nodes corresponded to those observed on TCL-SPION-enhanced MR images. TCL-SPION-enhanced MRI is useful for the detection and estimation of lymph node metastasis.