The effect of buttermilk powder addition post-curd formation or buttermilk addition to cheese milk on total and individual phospholipid content, chemical composition, enzyme activity, microbial populations and microstructure within Cheddar-style cheese was investigated. Buttermilk or buttermilk powder addition resulted in significant increases in total phospholipid content and their distribution throughout the cheese matrix. Addition of 10% buttermilk powder resulted in higher phospholipid content, moisture, pH and salt in moisture levels, and lower fat, fat in dry matter, L. helveticus and non-starter bacteria levels in cheeses. Buttermilk powder inclusion resulted in lower pH 4.6/Soluble Nitrogen (SN) levels and significantly lower free amino acid levels in 10% buttermilk powder cheeses. Buttermilk addition provided a more porous cheese microstructure with greater fat globule coalescence and increased free fat pools, while also increasing moisture and decreasing protein, fat and pH levels. Addition of buttermilk in liquid or powdered form offers potential for new cheeses with associated health benefits. © 2017 Elsevier Ltd