L-carnitine (LC) is an antioxidant with the ability to promote the growth in vitro embryo. OBJECTIVE The goal was to evaluate the effect of LC on some indicators of embryo development and blastocyst quality including zona pellucid (ZP) thickness, the hatching of blastocysts and their cell numbers. METHODS Mouse embryos were randomly divided into five groups and incubated with different concentrations of LC (I; 0, II; 0.5, III; 1, IV; 2 and V; 4 mg/ml) from 2-cell to hatched blastocyst. The percentage of blastocysts and hatched blastocysts was calculated. Blastocysts ZP thickness was measured and the number of blastocyst cells was counted using Hoechst and propidium iodide (PI) staining. RESULTS The results showed concentration of 0.5 mg/ml of LC had an antioxidant effect as in this group, the percentage of blastocysts and hatched blactocysts (p=0.01), the ZP thickness (p=0.00) and the number of blastocyst inner cell mass were significantly more favorable than the control group (p=0.03); and concentration of 4 mg/ml of LC had a toxic effect on embryo development and blastocyst quality (p=0.00). CONCLUSIONS The results suggest that LC may increase the number of blastocyst cells, which probably helps to expand the blastocyst and thinning of the ZP thickness and, therefore, creating a successful hatching for implantation.