Bioremediation of organophosphorus pesticides in contaminated foodstuffs using probiotics has been increasingly under the spotlight in recent years, though the biodegradation mechanism and derived intermediate products remain unclear. This study aimed to help fill this knowledge gap and examined the degradation mechanism of organophosphorus pesticide, chlorpyrifos, in milk by Lactobacillus delbrueckii ssp. bulgaricus using gas chromatography-tandem mass spectrometry (GC-MS/MS) combined with transcriptome analysis. After the strain was cultured for 20 h in the presence of chlorpyrifos, differential expressions of 383 genes were detected, including genes probably implicated during chlorpyrifos degradation such as those related to hydrolase, phosphoesterase, diphosphatase, oxidoreductase, dehydratase, as well as membrane transporters. GC-MS/MS analysis revealed the changes of secondary metabolites in L. bulgaricus during milk fermentation due to chlorpyrifos stress. 6-Methylhexahydro-2H-azepin-2-one, 2,6-dihydroxypyridine and methyl 2-aminooxy-4-methylpentanoate as intermediates, along with the proposed pathways, might be involved in chlorpyrifos biodegradation by L. bulgaricus.