Giardia intestinalis is a flagellated protozoan responsible for giardiosis (also called giardiasis in humans), the most prevalent and widespread parasitic infection in humans and mammals worldwide. The intestinal microbiota is highly diverse and any alteration in its composition may impact on the health of the host. While studies on the mouse model of giardiosis described the role of the gut microbiota in host susceptibility to infection by the parasite, little is known about the gut microbiota during natural infections in dogs and particularly in puppies. In this study, we monitored naturally G. intestinalis-infected puppies for 3 months and quantified cyst excretion every 2 weeks. All puppies remained subclinically infected during the sampling period as confirmed by fecal examination. In parallel, we performed 16S Illumina sequencing of fecal samples from the different time points to assess the impact of G. intestinalis infection on gut microbiota development of the puppies, as well as gut health markers of immunity such as fecal IgA and calprotectin. Sequencing results revealed that the canine fecal microbiota of Giardia-infected puppies becomes more complex and less diverse with increasing age. In addition, significant differences in the structure of the microbiota were observed between puppies with high and low Giardia cyst excretion. Chronic subclinical G. intestinalis infection appears to be associated with some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species and opportunistic pathogens, as well as a reduction of Lactobacillus johnsonii at specific time points. Calprotectin levels increased with age, suggesting the establishment of chronic low-grade inflammation in puppies. Further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).