Tartrazine (TZ) is a synthetic azo dye extensively used as a food colorant, posing potential harm to human health. This study examined whether maternal exposure to TZ at an acceptable daily intake (ADI) dosage during lactation could induce neurotoxicity in male rat pups and to investigate the potential underlying mechanism. Twelve rat dams at postnatal day 2 (PND2) were split equally into control (received vehicle), and TZ-treated (received TZ 7.5 mg/kg) groups.Administration was through oral gavage, once daily for 20 days (from PND2 to PND21). The pups’ exposure to TZ was via breastfeeding. On PND22, male pups’ brain tissues were collected for histopathological, immunohistochemical, biochemical, and gene expression analyses. Maternal TZ exposure during lactation led to brain tissue damage in rat pups, with resultant neuronal atrophy, nuclear condensation, perineuronal halos, capillary congestion, wide pericapillary spacing, hemorrhage, and neuropil vacuolation in the prefrontal cortex, cerebellar cortex, and hippocampus. Also, lactational TZ exposure was associated with oxidative stress (raised malondialdehyde, reactive oxygen species with total antioxidant capacity decline);oxidative DNA damage (overexpression of 8-OHdG protein); endoplasmic reticulum stress (upregulated XBP-1, BIP, CHOP, and JNK genes); autophagy suppression (upregulated p62 gene, downregulated Beclin-1 gene, decreased LC3-II at both protein and gene levels); synaptogenesis impairment (decreased synaptophysin protein expression and increased acetylcholinesterase activity level); inflammation (raised TNF-α, IL-1β, IL-6 with IL-10 decline). In conclusion, this study highlights neurotoxic alterations in pups associated with low-dose TZ exposure through breastfeeding, focusing on the underlying molecular mechanisms. This is crucial for developing strategies to safeguard public health.