Background: The benefits of the addition of L-carnitine to the diet of cats to control weight gain after the removal of their ovaries have been highlighted recently. It is also used in women after menopause. Whether its supplementation alters the sexual hormones is a concern. Objectives: The objective of this study was to evaluate the effect of dietary supplementation of L-carnitine on estrogen, progesterone and testosterone in cats following ovariohysterectomy. Methods: Conventional midline ovariohysterectomy was performed in 14 DSH adult and healthy female cats. 10 days after the surgery, the cats were divided into two groups randomly. In group 2, a single dose of 100 mg/kg/day L-carnitine was added to the regular diet for 50 days. The cats in group 1 received no L-carnitine in their diet. Blood samples were taken from all cats prior to surgery, and then again 10 and 60 days post-operatively. Serum levels of estrogen, progesterone and testosterone were measured using a commercially available AMH enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of estrogen and progesterone significantly decreased at 10 days after the surgery in all cats (P0.05). Conclusions: It is concluded that dietary supplementation of L-carnitine has no effect on serum levels of estrogen, progesterone and testosterone in cats following ovariohysterectomy. Long-term study is recommended.