We investigated in this study whether fluorescence imaging with a fluorophore-conjugated anti-human epidermal growth factor receptor 2 (HER2)/neu probe could be used to differentiate metastatic lymph nodes (LN) from normal LN to guide surgical resection. A fluorescent probe for detecting HER2/neu-expressing cells was generated by conjugation of the humanized anti-HER2/neu antibody trastuzumab with rhodamine. The green fluorescent protein-expressing breast cancer cell line 4T1-GFP was used for in vitro binding analysis and for the establishment of a model of HER2/neu expressing LN-metastatic breast cancer. All tumor-bearing mice were given a single intravenous injection of either rhodamine-conjugated anti-HER2/neu probe, or conjugated control IgG antibody, when LN metastasis developed. Each animal was imaged with both green and red fluorescence to assess in vivo binding of the rhodamine-conjugated anti-HER2/neu probe to the tumors and LN metastases. Hematoxylin and eosin (H&E) staining and immunohistochemistry was performed to confirm the presence of tumor and metastasis as well as HER2/neu expression. The imaging probe was able to bind to HER2/neu-expressing 4T1-GFP tumor cells in vitro. The primary tumors and LN metastases from the animals which were treated with rhodamine-conjugated anti-HER2/neu probe exhibited a visible red fluorescence signal. The fluorescent axillary LN metastasis was easily distinguishable from the surrounding normal tissue and normal LN. Sensitivity of 78% (14 out of 18) and specificity of 100% (20 out of 20) could be achieved with the rhodamine-conjugated anti-HER2/neu probe for the detection of LN metastasis. These data support further investigation of the fluorophore-conjugated anti-HER2/neu antibody to detect LN metastasis in the surgical setting.