Immediately after birth, mammals are largely colonized by microorganisms, with the gastrointestinal tract being the most commonly colonized organ. Over the years, several studies have shown that the intestinal microbiota is important for various physiological functions of the host. Gnotobiotic animal models are frequently used to better understand how the microbiota influences health and disease scenarios. Among gnotobiotic models, germ-free (GF) animals were first used in 1895, but it was not until 60 years later that germ-free colonies were suitable for large-scale experiments. The use of GF mice is an interesting and rich tool for studying the microbiome. However, their maintenance is a complex process that needs to be done carefully. In this chapter, we describe step by step how to manage and manipulate the gut microbiota of GF mice.