Acquired resistance to trastuzumab is a clinical problem in the treatment of HER2-over-expressing metastatic breast cancer. Importantly, an earlier report suggested that high body mass index was associated with reduced overall survival and reduced time to progression in patients with early stage or metastatic HER2-positive breast cancer treated with trastuzumab. Adipocyte-secreted factors may stimulate growth of HER2-positive cancers, blocking the growth inhibitory action of trastuzumab. Leptin and growth differentiation factor 15 (GDF15) are two adipocytokines that have been reported to stimulate HER2-PI3K signaling. We previously showed that cells with acquired trastuzumab resistance express increased levels of GDF15, and that GDF15 knockdown restores sensitivity to trastuzumab. The objective of the current study was to identify potential molecular mechanisms by which adipocytes stimulate resistance to trastuzumab in HER2-over-expressing breast cancer cell lines. Cells were cultured in complete media or conditioned media from differentiated adipocytes (CM). Cell viability of trastuzumab-treated cells was examined under anchorage-dependent and - independent conditions. Phosphorylation of Akt was assessed by Western blotting, and response to trastuzumab was reassessed upon treatment with the PI3K inhibitor LY294002 or after transfection with kinase-dead Akt. We report that CM significantly reduced trastuzumab-mediated growth inhibition of HER2-positive cells, and stimulated rapid phosphorylation of Akt. Pharmacologic or genetic inhibition of PI3K overcame CM-mediated trastuzumab resistance. Leptin and GDF15 were both measured in CM, but only GDF15 conferred resistance to trastuzumab. Leptin, on the other hand, abrogated sensitivity to lapatinib but not trastuzumab. Our observations suggest that adipocyte-secreted factors such as GDF15 stimulates PI3K signaling, resulting in reduced response to trastuzumab. The utility of adipocytokines as predictors of drug resistance and approaches to mitigate the cancer-promoting effects of adipocyte-secreted factors should be further examined. Our work supports additional investigation into GDF15 as a potential biomarker of trastuzumab resistance, and development of approaches to therapeutically target GDF15 in HER2-positive breast cancers that have progressed on trastuzumab.