There is an increased interest in investigating the relationship between the gut microbiota and energy homeostasis. Probiotics are health beneficial microbes mainly categorised under the genus Lactobacillus and Bifidobacterium, which when administered in adequate amounts confer health benefits to the host, and have been implicated in various physiological functions. The potential role of probiotics in energy homeostasis is a current and an emerging area of research. In the present study, Lactobacillus acidophilus NCDC 13 was used to evaluate its anti-obesity potential in diet-induced obese (C57BL/6) mice. The probiotic bacterial culture was administered in Indian yogurt preparation called ‘dahi’, prepared using native starter cultures, and compared with control dahi containing only dahi starter cultures. The dietary intervention was followed for 8 weeks, and whole-body fat composition, and liver and muscle adiposity were measured using MRI. Changes in gut microbiota were assessed by fluorescent in situ hybridisation in faeces and caecal contents. The feeding of the probiotic brought no changes in body-weight gain, food and dahi intake when compared with the control dahi-fed animals. No significant changes in body fat composition, liver and muscle adiposity were also observed. At the end of the dietary intervention, a significant increase (P < 0·05) in the number of total Bifidobacterium was observed in both faeces and caecal contents of mice as a result of probiotic dahi administration. Thus, L. acidophilus NCDC 13 supplementation could be beneficial in shifting the gut microbiota balance positively. However, its anti-obesity potential could not be established in the present study and warrants further exploration.