Camellia (Camellia oleifera Abel.) seed oil is a commonly used edible oil of China. In ancient Chinese literature, it is mentioned to be helpful for postpartum repair and lactation in women. Research on camellia seed oil (CO) as a feed additive for dairy cattle is less. We investigated the effect of CO on the expression of milk fat and protein syntheses-related genes in differentiated bovine mammary epithelial cells (MAC-T) using soybean oil (SO) as the control. The results showed that CO increased the expression of genes related to de novo synthesis of fatty acids including sterol regulatory element-binding protein 1 (SREBP1), acetyl-CoA carboxylase 1 (ACC), fatty acid synthase (FASN), lipoprotein lipase (LPL), and stearoyl-CoA desaturase (SCD) (p < .05). Among the milk protein genes analyzed, CO increased β-casein mRNA expression (p < .05) and decreased αS1-casein mRNA expression (p < .05) in MAC-T cells. CO upregulated the pathways related to milk protein synthesis with increased mRNA levels of phosphoinositide 3-kinase (PI3K), RAC-alpha serine/threonine-protein kinase (AKT1), and mammalian target of rapamycin (mTOR) (p < .05) in MAC-T cells. Ribosomal protein S6 kinase beta-1 (S6K1) gene was upregulated, and eukaryotic initiation factor 4E (eIF4E) gene (p < .05) was downregulated with CO treatment.The mRNA expression levels of janus kinase 2 (JAK2), activator of transcription 5-β (STAT5-β), and E74-like factor 5 (ELF5) were elevated in MAC-T cells treated with CO (p < .05). Meanwhile, the protein expression levels of S6K1, STAT5-β, phosphorylated mTOR (p-mTOR), p-S6K1, and p-STAT5-β increased in MAC-T cells treated with CO (p < .05). In summary, CO promoted β-casein synthesis by regulating PI3K-mTOR-S6K1 and JAK2-STAT5 signaling pathways and influenced fatty acid synthesis by regulating SREBP1-related genes in MAC-T cells. We need to further confirm the function of CO using in vivo models.