The mechanisms underlying the association between diabetes and inner ear dysfunction are not known yet. The aim of the present study is to evaluate the impact of obesity/insulin resistance on inner ear fluid homeostasis in vivo, and to investigate whether the organ of Corti could be a target tissue for insulin signaling using auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as an in vitro model. High fat diet (HFD) fed C57BL/6J mice were used as a model to study the impact of insulin resistance on the inner ear. In one study, 12 C57BL/6J mice were fed either control diet or HFD and the size of the inner ear endolymphatic fluid compartment (EFC) was measured after 30 days using MRI and gadolinium contrast as a read-out. In another study, the size of the inner ear EFC was evaluated in eight C57BL/6J mice both before and after HFD feeding, with the same techniques. HEI-OC1 auditory cells were used as a model to investigate insulin signaling in organ of Corti cells. HFD feeding induced an expansion of the EFC in C57BL/6J mice, a hallmark of inner ear dysfunction. Insulin also induced phosphorylation of protein kinase B (PKB/Akt) at Ser473, in a PI3-kinase-dependent manner. The phosphorylation of PKB was inhibited by isoproterenol and IBMX, a general phosphodiesterase (PDE) inhibitor. PDE1B, PDE4D and the insulin-sensitive PDE3B were found expressed and catalytically active in HEI-OC1 cells. Insulin decreased and AICAR, an activator of AMP-activated protein kinase, increased the phosphorylation at the inhibitory Ser79 of acetyl-CoA carboxylase, the rate-limiting enzyme in de novo lipogenesis. Furthermore, the activity of hormone-sensitive lipase, the rate-limiting enzyme in lipolysis, was detected in HEI-OC1 cells. The organ of Corti could be a target tissue for insulin action, and inner ear insulin resistance might contribute to the association between diabetes and inner ear dysfunction.