mTOR inhibitors such as everolimus and BEZ235 have demonstrated efficacy in pancreatic neuroendocrine tumors (PanNET) at the cost of severe side effects, and no biomarker currently predicts response. To identify molecular pathways of resistance, precision-cut slices of 17 fresh well-differentiated PanNET were cultured with everolimus or BEZ235 for 2 days and immunostained with cleaved caspase-3 and mTOR pathway markers. Transcriptomes of sensitive and resistant tumors were compared, and candidate pathways validated by immunohistochemistry. The predictive value of key proteins was then assessed in 26 PanNET patients treated with everolimus. mTOR inhibitors induced significant apoptosis and loss of pS6 and p4EBP1 in tumor slices, with 6/17 tumors considered sensitive. Transcriptomic analysis revealed that sensitive tumors displayed a mitochondrial-based metabolism, whereas resistant tumors exhibited a hypoxic and glycolytic profile, confirmed by high expression of CAIX, GLUT1, ATP5O, and mtTFA. Necrosis was absent and microvessel density similar in both groups, suggesting a pseudohypoxic metabolism in resistant tumors. High expression of CAIX and LDHA, two markers of pseudohypoxia/glycolysis, was associated with shorter progression-free survival in patients treated with everolimus. This study demonstrates that tissue culture can effectively assess drug response in PanNET and identifies a pseudohypoxic/glycolytic profile as a determinant of resistance to mTOR inhibition, detectable by immunohistochemistry and potentially noninvasively by ¹⁸F-FDG PET-CT.