Autocrine production of Insulin-like growth factor-1 (IGF-I) regulates growth of human intestinal muscle cells by activation of distinct PI 3-kinase-dependent and Erk1/2-dependent pathways. The aim of the present study was to determine the mechanisms by which IGF-I regulates G1 phase of the cell cycle and muscle cell proliferation. Incubation of quiescent cell with IGF-I stimulated time-dependent cell cycle progression measured using fluorescence activated cell sorting (FACS) analysis and incorporation of [³H]thymidine. Studies using a micro-array based approach were used initially to identify genes expressed in human intestinal muscle encoding proteins known to participate in the G1 phase of the cell cycle that were regulated by IGF-I. Incubation of muscle cells for 24 h with IGF-I elicited a greater than 5 fold increase in the expression of cyclin D1 (CCND1), and a greater than 2 fold increase in retinoblastoma protein (Rb1). IGF-I elicited a time-dependent increase in cyclin D1 protein levels that was meditated jointly by Erk1/2-dependent and PI 3-kinase mechanisms. The increase in cyclin D1 levels was accompanied by a time-dependent increase in cyclin D1-dependent, cyclin dependent kinase-4 (cdk4) activity. IGF-I also elicited a rapid time-dependent increase in Rb(Ser807/811) phosphorylation, the specific target of the cyclin D1-dependent cdk4 kinase, and a slower increase in total Rb protein levels. We conclude that IGF-I stimulates G1 phase progression, DNA synthesis and cell proliferation of human intestinal smooth muscle cells. The effects of IGF-I on proliferation are mediated jointly by Erk1/2-dependent and PI 3-kinase-dependent pathways that regulate cyclin D1 levels, cyclin D1-dependent cdk4 activity, and Rb activity.