Gut microbiota depletion from broad-spectrum antibiotic treatment reduces nocturnal sleep and locomotion in mice. Are these bugs playing a vital role in regulating sleep behavior? Insomnia could be nominated as one of the biggest hidden threats impairing the quality of life in many countries. According to a report from the American Academy of Sleep Medicine (AASM), insomnia has negative impacts on physical and mental health. It also impairs performance in studying and working and is the cause of numerous accidents and occupational errors. In the United States, insomnia is associated with approximately 253 million days of lost work each year, and the annual cost is estimated at more than $100 billion. This includes direct treatment expenses and indirect costs such as poorer workplace performance, increased risk of accidents and increased healthcare utilization1. Insomnia and sleep disorders may involve various factors. However, one to which we cannot turn a blind eye is antibiotic therapy. Since the accidental discovery of penicillin, antibiotics have been a miraculous and powerful weapon in the battle against bacterial infection. In the field of medicine, antibiotics are widely used around the globe as a general therapeutic method for many infectious diseases. In 2015, outpatient pharmacies in the U.S. issued 269 million antibiotic prescriptions, equivalent to five out of six people receiving one antibiotic prescription per year6. Nevertheless, antibiotic treatment can cause various adverse effects such as nausea, vomiting, diarrhea, and also insomnia. Evidence from a case report review has demonstrated that insomnia might be problematic for up to 13% of patients administered with only one of the fluoroquinolone drugs3. However, the mechanism underlying insomnia induced by antibiotic usage remains unknown. One thing is for sure: antibiotics destroy the bacterial community in the gut. Scientists have endeavored to explore and understand the relationship between the gut microbiome and the brain for years. Many studies have shown that the two may be closely related in a bidirectional way via the gut-brain axis2. The gut microbiome is defined merely as a collection of tiny bacteria in the gastrointestinal tract, which may be higher in number than all the cells of the body. They may also produce or secrete substances that regulate diurnal rhythms5 and influence social activities, stress responses, cognitive behaviors, and metabolism. Many studies have shown that germ-free mice with antibiotic-induced intestinal microbiota disruption have elevated locomotor activity, from which it is inferred that the sleep cycle may be affected7. Nonetheless, there is no tangible evidence to demonstrate a direct connection between gut microbiota and sleep behavior. Recently, Lendrum and colleagues4 attempted to prove this correlation by using ten germ-free mice divided equally into control and antibiotic treatment groups. After completion of 14 consecutive days of antibiotic regimens, fresh stools from both groups were collected and DNA was extracted to look for the presence of the bacterial 16s rRNA gene. This was used to study the diversity of the microbiome and confirm the depletion of microbes. The results demonstrated that mice treated with broad-spectrum antibiotics had greatly reduced gut microflora compared to the control group. In addition, the sleep cycles were recorded by using videosomnography immediately after completion of the antibiotic treatment with two types of video camera enabling continuous monitoring throughout a 24-hour cycle. This was divided into a light phase (0-12 hours) and a dark phase (12-24 hours). Subsamples of the video recordings were analyzed using the automated video-tracking software to study locomotor activity.