The microRNA-126 (miR-126) is a miRNA expressed in highly vascularized tissues, and it is believed to play a role in angiogenesis by repressing sprouty-related EVH1 domain containing 1 (Spred1). In the current study, we determined the expression pattern of chicken miR-126 (gga-miR-126) and predicted and validated its target genes. The quantitative reverse-transcription (qRT) PCR analysis showed that miR-126 was expressed in various chicken tissues with the highest level in lung. In liver, the expression level of miR-126 increased from 0 to 7 wk of age. The expression of miR-126 in primary chicken hepatocytes decreased with culturing. A miR-126 binding site was predicted in the 3' UTR (untranslated region) of chicken Spred1. Dual-luciferase reporter assays indicated that miR-126 could bind to the predicted site to repress the expression of Spred1. These data validate Spred1 as a target gene of chicken miR-126. These results will help further understand the function and regulation of miR-126 and Spred1 in chickens.