The aim of the current study was to explore the effect of pre-acidification and whey protein-based fat replacers (CH-4560 and YO-8075) on low-fat Mozzarella cheese (LFC). The color and browning, calcium, meltability, texutre and pizza bake performance of cheeses at 0, 7, 14, 21, 28 days storage was determined, and full-fat cheeses (FFC; milk containing 2.21 g fat/100 g milk) and LFC were manufactured as control. The LFC without fat replacer was harder, less sticky, and had high stretch force and low meltability, as well as poor pizza bake and stretch performance, while these appearance defects were partially corrected via the addition of whey protein concentrates and using pre-acidification. Pre-acidification of cheese milk to pH 5.5 or lower negatively affected the stickiness, and pizza bake, and stretch performance of LFC and LFC with fat replacers. As aging progressed, the meltability, stickiness and stretch performance of Mozzarella cheese improved, whereas the hardness and stretch force of Mozzarella cheese decreased. Our study will provide guidance for the preparation of higher quality low-fat mozzarella cheese.