Second-generation antipsychotics including olanzapine are associated with weight gain, dyslipidemia and other metabolic disorders. Both animal and clinical studies have shown that co-treatment with betahistine (a histamine H1 receptor agonist/H3 receptor antagonist) is effective in controlling olanzapine-induced weight gain. In the present study, we investigate whether co-treatment with betahistine is able to prevent dyslipidemia induced by chronic olanzapine treatment and the underlying mechanisms. Female rats were orally administered with olanzapine (1 mg/kg, t.i.d.) for 3.5 consecutive weeks and then a 2.5-week drug withdrawal. Then, rats were divided into 4 groups for 5 weeks treatment: (1) vehicle, (2) olanzapine-only (1 mg/kg, t.i.d.), (3) betahistine-only (9.6 mg/kg, t.i.d.), and (4) olanzapine and betahistine (O + B) co-treatment. After completing treatment, hepatic mRNA expression was measured by qRT-PCR, while the protein levels were detected by western blot. In our study, olanzapine-only treatment significantly increased triglyceride accumulation and non-esterified fatty acids (NEFA), and upregulated mRNA expression of sterol regulatory element binding protein 1 (SREBP-1) and its target genes, while these alterations were ameliorated by O + B co-treatment. Hepatic AMP-activated protein kinase α (AMPKα) was activated in the O + B co-treatment group, with a significant reduction in nuclear SREBP-1 protein expression but an increased expression of peroxisome proliferator-activated receptor-α (PPARα) and its-responsive molecule(CPT1A), compared with olanzapine-only treatment. In addition, olanzapine significantly increased hepatic histamine H1 receptors, while O + B co-treatment significantly reversed them to normal levels. This study provided the first evidence that betahistine could act on hepatic H1 receptors via modulation of AMPKα-SREBP-1 and PPARα-dependent pathways to ameliorate olanzapine-induced dyslipidemia in rats.