The objective of this study was to investigate the effects of tributyrin supplementation on liver fat metabolism in broiler chickens. Two hundred and forty broilers were randomly allocated into two experimental groups (6 replicates per treatment; 20 chickens in each replicate): the control group (CN), which received a basal diet, and the tributyrin group (TB), which received a basal diet supplemented with 1 g/kg of tributyrin. The experimental period lasted 37 days. The results showed that in the liver, broilers supplemented with tributyrin had higher content of high-density lipoprotein cholesterol (HDL-C) (p < 0.05). Liver hepatic lipase (HL), lipoprotein lipase (LPL) and total lipid (TL) activity were significantly lower than in the TB group than that in the NC group. Meanwhile, the diet supplemented with tributyrin had more lipid droplets than the NC group, whereas the TB and NC groups showed no histological abnormalities in the liver. Furthermore, the mRNA expression levels of peroxisome proliferators-activated receptor α (PPARα), proliferators-activated receptor γ (PPARγ), fatty acid synthase (FAS), LPL and adipose triglyceride lipase (ATGL) in the liver were significantly upregulated in the TB group (p < 0.05), while those of the long-chain acyl-CoA-synthetase 1 (ACSL1) mRNA between the TB group and the NC group were not different (p > 0.05). These findings indicated that the diet supplemented with tributyrin could increase fat deposition appropriately by promoting fat synthesis without causing liver tissue damage, which demonstrated that tributyrin can be considered a valid feed additive for broiler chickens.