As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3 gamma has been the subject of recent research. Nevertheless, whether 14-3-3 gamma can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3 gamma in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3 gamma overexpression significantly inhibited the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3 gamma overexpression also suppressed the production of TNF-alpha and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3 gamma overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3 gamma overexpression promoted the secretion of triglycerides and lactose and the synthesis of beta-casein. Furthermore, the expression of genes relevant to nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3 gamma overexpression inactivated the NF-kappa B and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-B (IB) phosphorylation levels, as well as by inhibiting NF-kappa B translocation. Meanwhile, 14-3-3 gamma overexpression enhanced the expression levels of beta-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR). These results suggest that 14-3-3 gamma was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-B and MAPK signaling pathways and up-regulatingm TOR signaling pathways to protect against LPS-induced injury.