Ovarian cancer, the most lethal gynaecological cancer, is characterised by the shedding of epithelial cells from the ovarian surface, followed by metastasis and implantation onto the peritoneal surfaces of abdominal organs. Our proteomic studies investigating the interactions between peritoneal (LP-9) and ovarian cancer (OVCAR-5) cells found transketolase (TKT) to be regulated in the co-culture system. This study characterized TKT expression in advanced stage (III/IV) serous ovarian cancers (n = 125 primary and n = 54 peritoneal metastases), normal ovaries (n = 6) and benign serous cystadenomas (n = 10) by immunohistochemistry. In addition, we also evaluated the function of TKT in ovarian cancer cells in vitro. Nuclear TKT was present in all primary serous ovarian cancer tissues examined (median 82.0 %, range 16.5-100 %) and was significantly increased in peritoneal metastases compared with matching primary cancers (P = 0.01, Wilcoxon Rank test). Kaplan-Meier survival and Cox regression analyses showed that high nuclear TKT positivity in peritoneal metastases (>94 %) was significantly associated with reduced overall survival (P = 0.006) and a 2.8 fold increased risk of ovarian cancer death (95 % CI 1.29-5.90, P = 0.009). Knockdown of TKT by siRNAs significantly reduced SKOV-3 cell proliferation but had no effect on their motility or invasion. Oxythiamine, an inhibitor of TKT activity, significantly inhibited the proliferation of four ovarian cancer cell lines (OV-90, SKOV-3, OVCAR-3 and OVCAR-5) and primary serous ovarian cancer cells isolated from patient ascites. In conclusion, these findings indicate that TKT plays an important role in the proliferation of metastatic ovarian cancer cells and could be used as novel therapeutic target for advanced disease.