Pancreatic β-cell mitogenesis is increased by insulin-like growth factor I (IGF-I) in a glucose-dependent manner. In this study it was found that alternative β-cell nutrient fuels to glucose, pyruvate, and glutamine/leucine independently induced and provided a platform for IGF-I to induce INS-1 cell DNA synthesis in the absence of serum. In contrast, long chain FFA (≥C₁₂) inhibited 15 mm glucose-induced [³H]thymidine incorporation (±10 nm IGF-I) by 95% or more within 24 h above 0.2 mm FFA complexed to 1% BSA (K_0.5 for palmitate/1% BSA = 65–85 μm for 24 h; t_0.5 for 0.2 mm palmitate/1% BSA = ∼6 h). FFA-mediated inhibition of glucose/IGF-I-inducedβ -cell DNA synthesis was reversible, and FFA oxidation did not appear to be required, nor did FFA interfere with glucose metabolism in INS-1 cells. An examination of mitogenic signal transduction pathways in INS-1 cells revealed that glucose/IGF-I induction of early signaling elements in SH2-containing protein (Shc)- and insulin receptor substrate-1/2-mediated pathways leading to downstream mitogen-activated protein kinase and phosphoinositol 3′-kinase activation, were unaffected by FFA. However, glucose-/IGF-I-induced activation of protein kinase B (PKB) was significantly inhibited, and protein kinase Cζ was chronically activated by FFA. It is possible that FFA-mediated inhibition of β-cell mitogenesis contributes to the reduction of β-cell mass and the subsequent failure to compensate for peripheral insulin resistance in vivo that is key to the pathogenesis of obesity-linked diabetes.