Abstract The gastrointestinal (GI) tract of a neonatal calf undergoes morphological and functional adaptations after birth. The GI tract during this period is influenced by many factors, including bioactive compounds in colostrum. Early colonization of the neonate gut microbiome can fundamentally influence the neonate predisposition to develop immune and metabolic disorders. Fecal microbiome transplantation (FMT) has demonstrated positive effects on the treatment of GI diseases in humans; therefore, we hypothesized that neonatal gut inoculation with a healthy adult gut microbiota via milk replacer can potentially improve gut development and maturation early in life. We aimed to evaluate the effects of performing an early life FMT in neonatal dairy calves using stool samples from on-farm selected adult healthy donors on growth and health performance, blood immunometabolites, and gene expression in immune cells [polymorphonuclear leukocytes cells (PMNL)]. The on-farm selection of the adult donor was based on health and production records at the Dairy Research and Training Facility (DRTF) at South Dakota State University, as well as fecal samples testing negative for Mycobacterium paratuberculosis, Salmonella, and Cryptosporidium. Sixteen healthy newborn Holstein calves (n = 8/trt) housed in individual hutches were used in a randomized complete block design from birth to 7 wk of age. Calves were fed 2.8 L/d of antibiotic-free milk replacer 2×/d during wk 1 to 5, 1×/d on wk 6, and weaned at d 42. Antibiotic-free starter pellets and water were fed ad libitum. Calves were assigned to either a baseline nutritional program (CON) or 1×/d inoculations with 25 g of fecal donor material (FMT) mixed in the milk replacer from 8 to 12 d of age. Individual intakes of milk and pellets were measured daily. Fecal and respiratory scores were recorded daily. Body weight (BW) and withers height (WH) were recorded weekly. Blood samples were collected weekly for immunometabolic profiling and PMNL isolation for gene expression analysis. Data were analyzed using the MIXED procedure of SAS. There was a trend (P = 0.09) for greater BW (50.8 vs 52.7 kg±0.7) in FMT calves. Similarly, there was a trend for greater WH (P = 0.13) in FMT (82.6 vs 83.8 kg±0.49) calves. Starter intake and fecal scores were not affected (P > 0.23) by FMT inoculation. Improvements observed in growth parameters by FMT inoculation are suggestive that neonatal dairy calves may benefit from this approach to enhance gut health and immunity, which might be further explained by blood immunometabolites and PMNL gene expression analysis.