Background: Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by persistent difficulties in social communication and interaction together with restricted or repetitive patterns of behavior. Aim: A study of changes in total carnitine levels was associated with clinical improvement after oral carnitine supplementation in children with autism spectrum disorder. Methodology: 120 treatment-naive children aged 3–7 years with Childhood Autism Rating Scale (CARS) scores of 35–50 were allocated equally to L-carnitine syrup (50 mg/kg/day in two divided doses) or matching placebo for six months. Results: CARS and serum total carnitine were assessed at baseline and month 6, and all participants completed follow-up. CARS decreased from 40.83±5.19 to 34.05±5.45 in the L-carnitine group and from 41.12±4.14 to 39.85±4.29 with placebo. The between-group difference in change was −5.52 points (95% CI −6.72 to −4.31; p<0.001; Cohen’s d = −1.66). Serum total carnitine increased from 29.10±9.25 to 50.22±7.24 µmol/L with L-carnitine but remained unchanged with placebo (28.82±6.20 to 28.98±6.19 µmol/L); the between-group difference in change was 20.96 µmol/L (95% CI 19.03 to 22.89; p<0.001). Within the active group, greater carnitine elevation was strongly associated with greater CARS reduction (Pearson r= −0.86, p<0.001). A predefined response of at least a 4-point CARS reduction occurred in 73.3% versus 5.0% of participants. Adverse events were mild and no participant discontinued treatment. Conclusion: Six months of L-carnitine improved serum total carnitine and autism severity relative to placebo, with a strong exposure-response association. Larger multicenter trials are required.