To investigate the length of lymphangiogenesis in the rectal tissue distal to a rectal cancer and its effect on the resection margins of the rectum, 63 specimens of normal rectal tissue distal to the tumor were collected from the surgical resections of ten rectal cancer patients. The specimens were taken at 0.5-cm intervals between the distal end of tumor and the distal surgical margin. The mean amount of collected tissue in each patient was 6.3. The expression of VEGFR-3 and Prox1 was measured in the specimens using real-time quantitative reverse transcription polymerase chain reaction. VEGFR-3 and Prox1 were expressed in all harvested tissues. There was a reduction of expression of VEGFR-3 (nine cases) and Prox1 (ten cases) from tissue adjacent to the rectal tumor to the distal resection margin of the rectum. A downward slope of the expression levels of VEGFR-3 and Prox1 in all cases was found at less than 3.0 cm distal to the tumor. The median VEGFR-3 expression level in the tissues within 1.5 cm adjacent to the rectal cancer was higher than in those tissues beyond 1.5 cm (P = 0.024). The median Prox1 expression level in the tissues within 1.0 cm distal to the rectal cancer was higher than in those tissues beyond 1.0 cm (P = 0.003). Lymphangiogenesis may facilitate the mural spread of cancer cells. The length of development of new lymphatics in the rectal wall distal to a rectal cancer may be less than 3.0 cm. Resection of a rectal cancer should routinely include 3.0 cm distal to the tumor to ensure adequate excision of tissue subject to lymphangiogenesis and potential mural spread of the tumor.