PM2.5, recognized as a potential pathogenic factor for nervous system diseases, remains an area with many unknowns, particularly regarding its effects on human health. After five-month real-ambient PM2.5 exposure, we observed no significant pathological damage to the lung, liver, spleen, or kidney tissues. However, PM2.5 exposure led to neuronal degeneration in the hippocampal CA1 region of Brown Norway (BN) rats. The level of IL-6, IL-13, IL-1β, IL-12, IL-4, GRO/KC, MIP-1α, CM-CSF significantly increased in lung lavage fluid (P < 0.05 for all). Notably, we detected a slight impairment in spatial learning ability, as evidenced by the Barnes maze training outcomes. There were no significant changes in the bacterial community in lung lavage fluid (P = 0.621), but the bacterial community in the gut significantly changed (P < 0.001), with more species identified (P < 0.05). The metabolomic analysis revealed 147 and 149 significantly changed metabolites in the pulmonary system and serum, respectively (P < 0.05). PM2.5 exposure caused a decrease in Nervonic acid (NA) in both the lung and serum, which likely contributed to spatial learning impairment (P < 0.01). The correlation between lung metabolites, gut bacterial species, and serum metabolites indicated that PM2.5 exposure likely impaired spatial learning through the lung-gut-brain axis pathway. Lung and serum metabolic disorders and intestinal microbial imbalance occurred in BN rats post-five-month real-ambient PM2.5 exposure. There were two potential ways that PM2.5 exposure caused the decline of spatial learning ability in wild-type BN rats: (1) PM2.5 exposure led to a significant decrease of neuroprotective Nervonic acid in lung and serum metabolites. (2) PM2.5 exposure likely led to reduced spatial learning ability through the lung-gut-brain axis.