Fatty liver hemorrhagic syndrome (FLHS) is a metabolic condition of chicken and other birds caused by diverse nutritional, hormonal, environmental, and metabolic factors. Here we studied the effect of different diet composition on the induction of FLHS in single comb White Leghorn (WL) Hy-line laying hens. Seventy six (76) young WL (26 wks old) laying hens and 69 old hens (84 wks old) of the same breed were each divided into 4 treatment groups and provided 4 different diet treatments. The diet treatments included: control (C), 17.5% CP, 3.5% fat (F); normal protein, high fat (HF), 17.5% CP, 7% F; low protein, normal fat (LP), 13% CP, 3.5% F; and low protein, high fat (LPHF), 13% CP, 6.5% F. The diets containing high fat also had a higher ME of 3,000 kcal/kg of feed while the other 2 diets with normal fat had a regular lower amount of ME (2750 kcal/kg). Hen-day egg production (HDEP), ADFI, BW, egg weight, plasma enzymes indicating liver damage (alkaline phosphatase [ALP], aspartate aminotransferase [AST], gamma-glutamyl transferase [GGT]), liver and abdominal fat weight, liver color score (LCS), liver hemorrhagic score (LHS), liver fat content (LFC), liver histological examination, lipid peroxidation product in the liver, and genes indicating liver inflammation were evaluated. HDEP, ADFI, BW, and egg weight were significantly decreased in the LPHF diet group, while egg weight was also decreased in the LP diet group. In the young hens (LPHF group), ALP was found significantly higher at 30 d of diet treatment and was numerically higher throughout the experiment, while AST was significantly higher at 105 d of treatment. LCS, LHS, and LFC were significantly higher in young hens on the LPHF diet treatment. A liver histological examination shows more lipid vacuolization in the LPHF treatment diet. HF or LP alone had no significant effect on LFC, LHS, or LCS. We suggest that LP in the diet with higher ME from fat can be a possible natural cause for predisposing laying hens to FLHS.