Lactoferrin (LF) is secreted by mammals and displays extensive biological effects. We previously reported that bovine LF (BLF) can boost the cold tolerance of a well-applied probiotic strain, Lactobacillus rhamnosus GG (LGG), to grow robustly under a cold environment, but the molecular mechanism is not clear. Here, RNA-seq analysis was conducted to ascertain molecular pathways underlying cold tolerance exerted by BLF. LGG was cultured in a cold environment (22 °C) in the presence or absence of BLF. Transcriptome analysis indicated that BLF significantly elicited 1.2-3.2 fold (log2 Fold change) higher expression levels of genes related to stress, defense, cell division, and transporter in LGG, including the genes CspA, LytR, XRE, MerR, and GpsB. The KEGG pathway and GO analyses confirmed that BLF can modulate a few central pathways to boost the growth of LGG. BLF also reduced metabolic pathways involved in purine, amino acid, pyrimidine, one-carbon metabolism, and secondary metabolites in LGG. We speculate that the reduction of the above pathways may play key roles to reduce energy requirement and maintain carbon metabolism balance in LGG for surviving and growing in a cold state, and BLF can be an excellent prebiotic to LGG cultured in this cold condition (22 °C). Overall, this study uncovers the molecular effects of BLF on LGG.