Abstract Galactooligosaccharide (GOS) is a prebiotic containing beta-linked galactose oligomers of 2 to 8 units. Previously, GOS positively impacted post-weaning growth performance and altered jejunal morphology. In this study, GOS-enriched whey permeate (Milk Specialties Global, Eden Prairie MN) was supplemented to piglets in farrowing and nursery phases. To maximize pre-weaning GOS intake, novel gruel creep feeders were used. Piglets from 24 litters received either no creep feed (NC), creep without GOS (CG-) or creep with 5.0% GOS (CG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). At d 22 (pre-weaning) and d 31 (post-weaning), 6 pigs per treatment were euthanized for sample collection. DNA was extracted from cecal and rectal swabs for downstream 16S rRNA sequencing via Illumina MiSeq (Zymo Research Corp, Irvine CA). An amplicon sequence variant (ASV) table was created using the DADA2 pipeline in R and taxonomy assignment was performed using the SILVA database. Average sequencing depth after filtering was 138,323 and low sequencing counts were discarded based on 20% prevalence. Data were analyzed using the lmer and adonis procedures in RStudio ver. 4.2.2. Analysis of 16S rRNA genes did not detect pre- or post-weaning GOS effects on Chao1, Simpson, or Shannon alpha diversity measures (P > 0.1). Similarly, no pre-weaning GOS effects were detected in Principal Coordinates Analysis (PCoA) beta diversity plots (P > 0.1); however, pigs fed GOS post-weaning segregated from controls (P < 0.01). The relative abundance of several genera including Fusicatenbacter and Collinsella increased by 2.0 and 1.3-fold, respectively, in the cecum of NG+ pigs, whereas, Bacteroides decreased by 3.8-fold (FDR, P < 0.05). In the rectum, relative abundance of Corprococcus decreased by 2.0-fold and Fusicatenbacter increased by 1.5-fold in NG+ pigs compared with NG- pigs (FDR, P < 0.05). Collectively, these genera in the cecum and rectum comprise of less than 3% of the total community. In contrast, lactobacillus in the cecum (33%) and rectum (21%) had a large relative abundance but was unaffected by treatment. We conclude that supplementation of GOS in the post-weaning phase 1 nursery diet alters the hindgut microbial community and may contribute to improvements in growth performance.