Lactococcus lactis subsp. lactis biovar diacetylactis is widely used in dairy fermentations as it can form the butter aroma compounds acetoin and diacetyl from citrate in milk. Here we explore the possibility of producing acetoin from the more abundant lactose. Starting from a dairy isolate of L. lactis biovar diacetylactis, we obtained a series of mutants with low lactate dehydrogenase (ldh) activity. One isolate, RD1M5, only had a single insertion mutation in the ldh gene compared to its patental strain through whole genome re-sequencing analysis. We tested the ability of RD1M5 to produce acetoin in milk. With aeration all the lactose could be consumed, and the only product was acetoin. In a simulated cheese fermentation, a 50% increase in acetoin concentration could be achieved. RD1M5 turned out to be an excellent cell factory for acetoin and was able to convert lactose in dairy waste into acetoin with high titer (41 g/L) and high yield (above 90% of the theoretical yield). Summing up, RD1M5 was found to be highly robust, and to grow excellently in milk or dairy waste. Being natural in origin opens up for applications within dairies as well as for safe production of food-grade acetoin from low-cost substrates.