This study was conducted to evaluate the effects of Lactobacillus acidophilus supplementation in different energy and nutrient density diets on growth performance, nutrient digestibility, blood characteristics (blood urea nitrogen and creatinine), fecal microbiota shedding, and fecal noxious gas emission in weaning pigs. A total of 140 crossed [(Landrace×Yorkshire)×Duroc] weaning pigs with an initial body weight (BW) of 7.51±0.79kg were used in a 42-day trial. Pigs were randomly allocated into one of four treatment groups in a 2×2 factorial arrangement with two levels of energy and nutrient density (Phase1: 3750 and 3900kcal/kg; Phase2: 3550 and 3700kcal/kg) and L. acidophilus (0 and 0.1%) according to sex and BW (7 replicates each with 2 gilts and 3 barrows). Pigs fed high energy and nutrient density diets increased (P<0.05) average daily gain (ADG), gain:feed (G:F) ratio, as well as H2S and acetic acid emission than those fed low energy and nutrient density diets. Pigs fed L. acidophilus supplementation diets increased ADG and G:F (P<0.05) compared with pigs fed diets without L. acidophilus supplementation. Moreover, L. acidophilus supplementation led to a significant (P<0.05) increase in DM digestibility on day 14 and shift microbiota by increasing fecal Lactobacillus, while decreasing E.coli counts, as well as a significant (P<0.05) decrease in serum BUN concentration and fecal noxious gas emission. The interactive effects (P<0.05) of dietary energy and nutrient density and L. acidophilus supplementation were observed on the fecal H2S and acetic acid gases emission. In conclusion, dietary supplementation of L. acidophilus improved growth performance, ATTD of DM, shifted microbiota by increasing fecal Lactobacillus and decreased E. coli counts, decreased BUN concentration and fecal noxious gas emission in weaning pigs. No interactive effects of L. acidophilus supplementation and dietary energy and nutrient density on growth performance, nutrient digestibility (DM, N, and GE), blood characteristics (BUN and creatinine) and microbiota (Lactobacillus and E. coli). While the beneficial effects of L. acidophilus supplementation on H2S and acetic acid gases emission are more dramatic with high energy and nutrient density diets.