Ocimum gratissimum and Ocimum basilicum are plants ethnopharmacologically used to treat diabetes mellitus, a life-threatening disease that affects millions of people worldwide. In order to further understand their antidiabetic potential, which has been previously demonstrated in animal models, we aimed to investigate the acute and chronic effects of major phenolic substances from both plants on insulin secretion and gene expression in pancreatic islets isolated from NMRI mice. Insulin secretion was measured after acute (1 h) and long-term (72 h) incubation of islets with one of four cinnamic acid derivatives (caftaric, caffeic, chicoric, and rosmarinic acids) or a C-glucosylated flavonoid (vicenin-2). All substances acutely enhanced glucose-stimulated insulin secretion (GSIS) from islets at concentrations from 10-10 to 10-6 M. They also increased GSIS after chronic incubation (10-8 M). None of them increased insulin secretion in the presence of low glucose concentration. Furthermore, these substances markedly changed the gene expression profile of key insulin regulatory genes INS1, INS2, PDX1, INSR, IRS1, and proliferative genes as well as glucose transporter 2 (GLUT2), in treated islets. Thus, they may play an important role in diabetes treatment. This is the first report on the insulin-secretory activity of caftaric acid, rosmarinic acid, and vicenin-2.