Consumption of raw milk may pose a health threat due to a possible contamination with certain pathogens. Thermal processing can guarantee microbial safety, but this procedure meanwhile alters the physicochemical property of milk and denatures original bioactive components. To assess whether these changes could lead to diverse functional consequences, 4 types of bovine milk (raw, pasteurized, UHT sterilized, and in-can sterilized) were digested in vitro and then applied to culture human intestinal Caco-2 cells. Subsequent transcriptomic analysis demonstrated that the genome expressive patterns of cells under the condition of raw milk and pasteurised milk were quite similar, definitely distinct from UHT and in-can sterilized group. Compared with raw milk group, 3, 662, and 1,904 differentially expressed genes (DEG, absolute fold change >1.5 and false discovery rate <0.05) were identified in pasteurized, UHT, and in-can milk culturing cells, respectively. Gene ontology analysis using overlapping DEG in UHT and in-can group showed that the most significantly enriched biological processes were allocated to cell cycle classes. A key gene CDK1, which controls the transition of G₂/M phase, was downregulated in both sterilized milk culturing cells. Moreover, some important genes (NRAS, KRAS, PI3KR, PIK3C3, RHEB, EIF4EBP1, EIF4E) involved in PI3K-AKT-mTOR-EIF4EBP1 pathway were deregulated, which together declined protein translation and may eventually cause cell cycle arrest. As many previous studies reported that the gross nutritional value of milk can be reduced by heat treatment, its underlying mechanisms were further illustrated by the transcriptomic profiling in this work.