MicroRNAs play extensive roles in cellular development. Analysis of the microRNA expression pattern during intestinal cell proliferation in early life is likely to unravel molecular mechanisms behind intestinal development and have implications for therapeutic intervention. In this study, we isolated mouse intestinal crypt cells, examined the differences in microRNA expression upon IGF-1 stimulated proliferation and identified miR-103 as a one of the key regulators. Mouse intestinal crypt cells were cultured and treated with IGF-1 for 24 h. MicroRNA microarray showed that multiple microRNAs are regulated by IGF-1, and miR-103 was the most sharply down-regulated. Expression of miR-103 in mouse intestinal crypt cells was confirmed by real-time Q-PCR. Sequence analyses showed that, among the 1040 predicted miR-103 target genes, CCNE1, CDK2, and CREB1 contain complementary sequences to the miR-103 seed region that are conserved between human and mouse. We further demonstrated that miR-103 controls the expression level of these three genes in mouse crypt cells by luciferase assay and immunoblotting assay. Taken together, our data suggest that in mouse intestinal crypt cells, miR-103 is part of the G1/S transition regulatory network, which targets CCNE1, CDK2, and CREB1 during IGF-1 stimulated proliferation.