Probiotic therapy targeting gut-brain axis has been proven to be effective in treating autistic patients. The present study aimed to assess the ability of three Lactobacillus strains (L. helveticus CCFM1076, L. acidophilus La28, and L. acidophilus JCM 1132) to alleviate autistic-like behavioral symptoms in VPA-treated rats from weaning to sexual maturation. For the first time, we assessed the synthesis of 5-hydroxytryptamine (5HT) and the metabolic capacity of the 5HT system in the peripheral and central nervous systems (PNS and CNS, respectively) based on tryptophan metabolism based on VPA-induced autism model. We also assessed gut microbiota, and short-chain fatty acids (SCFAs) at the end of week 8. While improving autistic-like behavioral symptoms, we found L. helveticus CCFM1076 was more beneficial in regulating 5HT anabolism and catabolism, balancing excitatory and inhibitory neurotransmitter release in the PNS and CNS, and increasing oxytocin (OT) synthesis in the hypothalamus. A significant correlation was noted between 5HT levels and the release of GABA, glutamate (Glu), and OT, suggesting that 5HT plays a vital role in the neuroendocrine network. Analyses of the gut microbiota and SCFA levels revealed greater Turicibacter abundance and lower butyric acid levels in VPA-treated rats, which have been reported to be associated with 5HT levels. L. helveticus CCFM1076 helped reduce Turicibacter abundance and up-regulate butyric acid levels, while L. acidophilus La28 and L. acidophilus JCM 1132 did not. L. helveticus CCFM1076 restored neurotransmitter homeostasis by improving the balance of the 5HT system in the PNS and CNS, thereby ameliorating autistic-like behaviors. This finding will help in the development of bioproducts for treating autism and in the establishment of a treatment model mimicking the intestinal environment of autistic patients.