The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy. Cancer Res; 72(10); 2683-91. Ó2012 AACR.