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We examined whether depth of subserosal cancer invasion predicts lymph node involvement and survival in gallbladder carcinoma (GBC) patients with pathologicial subserosal invasion (pT2), to explore which patients benefit from radical second resection among patients with inapparent pT2 tumor.Subjects comprised 31 patients with pT2 GBC. Thickness of the subserosal layer and vertical length of carcinoma invasion into the subserosa were measured under microscopy. Depth of subserosal invasion was divided subjectively into three categories: ss1, ss2, and ss3 (invasion of upper, middle, and lower thirds of the sub-serosal layer, respectively). Relationships between subserosal subclassification, histopathological factors, and prognosis were examined.Subserosal layers were significantly thicker (P < 0.001) in portions with cancer invasion (5.46 ± 0.68 mm; range 1.0 ∽ 13.75 mm) than those without cancer invasion (1.89 ± 0.16 mm, range, 0.88 ∽ 4.50 mm). Depth of carcinoma invasion into subserosa was 4.20 ± 0.65 mm (range, 0.25 ∽ 12.5 mm). Rate of lymphatic permeation, venous permeation, and lymph node involvement significantly increased with deeper subserosal invasion (P = 0.014, P = 0.027, P = 0.018, respectively). Among histopathological factors examined, only subserosal subclassification had a significant correlation with presence or absence of lymph node metastasis. Further, there was a significant correlation (P = 0.043) between the degree of subserosal invasion (ss1, ss2, and ss3) and involved nodal disease (pN0, pN1, and pM1 [lymph]). Although 5-year survival rates, according to the degree of subserosal invasion, tended to decrease with deeper invasion into the subserosal layer (ss1, 83.3%; ss2, 62.5%; ss3, 50.0%), no significant differences were noted.Pathological characteristics tend to become more aggressive with increasing depth of subserous carcinoma invasion in pT2 GBC. Depth of subserosal invasion is a predictor of presence and degree of lymph node metastasis in pT2 GBC. A sampling biopsy of the para-aortic nodes is recommended for inapparent pT2 GBC patients with subserosal invasion beyond one-thirds of the subserosal layer when they undergo radical second resection.