We showed that the rat Na⁺/P_i cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin-induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi-1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn¹⁷ Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway.