The viability and functional activity of lactic acid bacteria (LAB), such as Lactobacillus acidophilus in a fermented milk product is important. One of the functional activities of the LAB in fermented milk is the ability of the LAB to positively impact the human health. This study aimed to determine the effect of frozen storage on the fermented milk, namely acidophilus milk, based on the nutritional milk quality, the viability of L. acidophilus, and its potential to lower cholesterol levels in Wistar rats. The parameters measured was including milk quality (mainly pH, protein content, lactic acid levels, and syneresis), L. acidophilus bacterial viability in frozen storage (for 1, 2, and 3months), and some biological assays to evaluate the potential of milk in lowering blood cholesterol levels in Wistar rats. The result of this study suggests that acidophilus milk quality can be maintained in frozen storage for two months, and it had lactic acid levels of 1.07%, pH of 4.08, and protein levels of 3.33%. Giving acidophilus milk to Wistar rats for 15days could reduce the cholesterol level continuously until 30days of treatment. Therefore, this study proves that acidophilus milk quality can be maintained very well in frozen storage, and its functional properties to lower the cholesterol level of Wistar rats can be achieved after two weeks of consumption.