NLRP6 is a Nod-like receptor expressed in the intestinal epithelium. Previous studies reported a protective role for NLRP6 against intestinal injury and colitis-associated carcinogenesis via the regulation and establishment of a healthy microbiota. However, these results were not obtained using littermate animals, leaving the possibility that the pro-colitogenic microbiota phenotype associated with knockout (KO) mice was stochastically acquired and genotype independent. Here, we analyzed the microbiota at three intestinal locations from Nlrp6-/- and wild-type (WT) littermates, either co-caged or individually caged after weaning. Our results demonstrate that NLRP6 does not significantly influence the intestinal microbiota at homeostasis, and they support a previously reported sex-biased microbial community structure. Moreover, WT and Nlrp6-/- littermate mice displayed comparable sensitivity to dextran sulfate sodium (DSS)-induced colitis, although increased sensitivity was noted in KO females. Our results clarify the role of NLRP6 in microbiota and colitis control, and they highlight the importance of analyzing littermate animals in such studies.