Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. Unlike murine models, to which various standardized high lipid diets such as a high-cholesterol diet (HCD) are available, there has yet to be a uniformly adopted zebrasfish HCD protocol. In this study, we have developed an improved HCD protocol and thoroughly tested its impact on zebrafish lipid deposition and lipoprotein regulation in a dose- and time- dependent manner. The diet stability, reproducibility, and fish palatability were also validated. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae for 8 days produced hepatic steatosis that become more stable and severer after 1 day of fasting and was associated with an opaque liver phenotype (dark under transmitted light). Unlike larvae, adult fish fed HCD for 14 days followed by a 3 day fast did not develop a stable fatty liver phenotype, though the fish had higher ApoB-LP levels in plasma and an up-regulated lipogenesis gene fasn in adipose tissue. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.