The present study examined the direct effects of high glucose and insulin on protein synthesis in cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts. Cultured rat cardiac myocytes and fibroblasts were grown in media containing normal glucose, high glucose, or osmotic control, and incubated with or without insulin. In cardiac myocytes, high glucose had no effect, but insulin increased protein synthesis and atrial natriuretic peptide (ANP) secretion and gene expression. The extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor and the protein kinase C (PKC) inhibitor blocked insulin-induced protein synthesis. In cardiac fibroblasts, high glucose and osmotic control media increased DNA synthesis. Collagen synthesis and fibronectin and transforming growth factor-beta1 (TGF-beta1) mRNA expression were stimulated by high glucose, but not by osmotic control. Insulin increased DNA and collagen synthesis in fibroblasts, and the insulin-induced increase in DNA synthesis was blocked by the phosphatidylinositol 3 kinase (PI3K) inhibitor. Our findings suggest that cardiomyocyte protein synthesis is mainly regulated by insulin rather than high glucose and both high glucose and insulin contribute to fibroblast DNA and collagen synthesis. High glucose accelerates fibroblast DNA synthesis and collagen synthesis, and fibronectin and TGF-beta1 mRNA expression, dependent or independent of osmotic stress. Insulin regulates myocyte protein synthesis and fibroblast DNA synthesis through different intracellular mechanisms.