As the most abundant microRNA (miRNA) in the liver, miR-122 plays important roles in the growth and development of liver, lipid metabolism, and liver diseases. Vanin 1 (VNN1) plays an important role in hepatic lipid metabolism, and VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis. In our previous RNA-seq study, we found the expression of VNN1 increased significantly when the expression of miR-122 (gga-miR-122-5p) was knocked down in primary chicken hepatocytes. In this study, we verified this result by real-time qRT-PCR, and we also found that the chicken VNN1 was highly expressed in the liver. By bioinformatics analyses, we found the 3′UTR of VNN1 contained sequences completely complementary to the nucleotides 1 to 8 of miR-122. Co-transfection and dual-luciferase reporter assays showed that overexpression of miR-122 decreased the expression of luciferase reporter gene linked to the 3′UTR of chicken VNN1 in the Chinese hamster ovary cells (P < 0.01), and the decrease was further demonstrated to be dependent on the predicted miR-122 binding sites by site mutation analyses. These results further support miR-122 as a negative regulator of VNN1 expression in chicken hepatocytes. Overall, this study suggests that miR-122 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the VNN1 gene.