Abstract: Production of eggs of low cholesterol level is a prerequisite of modern studies to provide healthy food for human consumption. The present study was performed to assess the effect of canola oil (1 and 2%) on egg production, egg quality and biochemistry of plasma and egg yolk of laying hen. To achieve this goal, 75 hens (hisex, 26 weeks old) were divided into three treatments (25 hens for each) of 5 replicates (5 hens for each). In the first treatment, hens were fed basal diet free of canola whereas 1 and 2% canola oil were incorporated in the second and third treatment, respectively for eight weeks and all data were recorded on 4 and week 8 of the experiment. The current study revealed insignificant difference in feed intake, feed conversion ratio, egg production, egg weight, shell thickness, haugh units and egg yolk color among treatment groups. Plasma triacylglycerol and cholesterol values reduced significantly (p<0.05) in layers fed diet supplemented with canola oil after 8 weeks compare to control. Egg cholesterol values were reduced significantly (p<0.05) in eggs produced by hens supplemented with both concentrations of canola oil for 4 and 8 week compare to control. Liver and kidney function were not affected in all treatments throughout the study. Based on the current results, inclusions of 1% canola oil in layers diet were quiet enough to reduce plasma and egg cholesterol. These results open new perspectives on economy of poultry industry for production of healthy food (egg) for human consumption.