Fatty liver is a major metabolic disorder of dairy cows. One important reason is that hepatic very low-density lipoproteins (VLDL) assembly was significant decreased in dairy cows with fatty liver. In addition, the impairment of insulin-like growth factor (IGF)-1 synthesis was involved in the development of fatty liver. Therefore, the objective of this study was to investigate the effects of IGF-1 on the VLDL assembly in cow hepatocytes. In this study, cow hepatocytes were cultured and then transfected with Ad-GFP-IGF-1 (inhibited the IGF-1 expression) and Ad-GFP (negative control), and treated with different concentrations of IGF-1, respectively. The results showed that IGF-1 increased the mRNA abundance of apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), microsomal triglyceride transfer protein (MTTP), and low-density lipoprotein receptor (LDLR) and then increased the VLDL assembly in cow hepatocytes. Nevertheless, impairment of IGF-1 expression by Ad-GFP-IGF-1 could inhibit above genes expression and VLDL assembly in hepatocytes. Taken together, these results indicate that IGF-1 increases the VLDL assembly and impairment of IGF-1 expression decreases the VLDL assembly in cow hepatocytes.