Gluconeogenesis in renal proximal tubules (PTs) is markedly enhanced in prolonged fasting and even in diabetic patients, which is comparable to the liver. However, its pathophysiological significance and hormonal regulation are not well known. We have recently found that cAMP increases mRNA expression levels of gluconeogenic enzyme, and insulin suppresses PT gluconeogenesis via insulin receptor substrate (IRS)1/Akt2 pathway (ASN Kidney Week 2017). On the other hand, parathyroid hormone (PTH) has also been reported to enhance PT gluconeogenesis along with the fact that the high level of PTH is associated with abnormal glucose metabolism. Therefore, in this study we investigated the details of PTH-enhanced gluconeogenesis in PTs. PTs were freshly isolated from rat kidneys or human kidneys, which were surgically obtained from consented patients of kidney cancer. To analyze the direct effect and mechanism of PTH on PT gluconeogenesis, PTs treated with 10 µM PKA (protein kinase A) inhibitor H89 or 100 nM PKC (protein kinase C) inhibitor Gö6983 were incubated with 1 nM PTH in DMEM overnight. To examine the relationship to insulin signaling, PTs were stimulated by 10 nM insulin for 4-hr after overnight incubation with PTH or 0.2 mM cAMP. We also performed siRNA against FoxO1 (forkhead box protein O1). During the whole process, PT gluconeogenesis was evaluated by measuring the mRNA expression levels of gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6P) by quantitative PCR. The mRNA expression levels of FoxO1 were also measured by quantitative PCR. Further, we performed western blotting in both of rat and human kidney cortex to analyze Akt phosphorylation in insulin signaling pathway. In PTs, PTH significantly increased the mRNA expression levels of PEPCK and G6P by 6 and 4 times, respectively. Not PKA inhibitor but PKC inhibitor completely abolished this PTH effect, which was consistent with PKC-mediated PTH signaling pathway. Interestingly, insulin failed to inhibit this PTH-enhanced PT gluconeogenesis, while insulin completely inhibited cAMP-enhanced PT gluconeogenesis. Moreover, siRNA against FoxO1 completely dissipated the PTH effect. In the kidney cortex, insulin induced Akt phosphorylation, which was preserved after incubation with PTH. Our results, for the first time to our knowledge, revealed that PTH increases mRNA expression levels of gluconeogenic enzymes in PTs via PKC pathway, and induces insulin resistance in PTs. Considering these results, the high level PTH may cause poor glucose control in these states through the direct enhancement of PT gluconeogenesis or exacerbating insulin resistance.