Background The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation. Principal Findings Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk. Conclusion Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.