This study was conducted to evaluate the effects of Clostridium butyricum dietary supplementation on the growth, antioxidant, immune response, gut microbiota, and intestinal barrier function of broilers under high stocking density (HSD) stress. A total of 324 1-day-old Arbor Acres male broilers were randomly assigned to three treatments with six replicates, each replicate including 18 chickens (18 birds/m2). The experiment lasted 6 weeks. The three treatments were basal diet (control, CON), basal diet supplemented with 1 × 109 colony forming units (cfu)/kg C. butyricum (CB), and basal diet supplemented with 10 mg/kg virginiamycin (antibiotic, ANT). The results showed that the body weight (BW) and average daily gain (ADG) of broilers in the CB group were significantly higher than those in the CON group in three periods (p < 0.05). The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in serum of the CB group were significantly increased compared with those in the CON and ANT groups at 42 days (p < 0.05). At 42 days, the serum immunoglobulin M (IgM) and immunoglobulin G (IgG) levels of the CB group were significantly higher than those of the CON group. Compared with the CON group, interleukin-1β (IL-1β) in the CB group was significantly decreased in the starter and grower stages (p < 0.05), but there was no significant difference between the two treatment groups (p > 0.05). C. butyricum significantly decreased the high stocking density-induced expression levels of IL-1β and tumor necrosis factor-α (TNF-α) in the ileum of broilers at different stages. Additionally, C. butyricum could increase the expressions of claudin-1 and zonula occludens-1 (ZO-1) in intestinal tissue. Moreover, C. butyricum significantly increased the Sobs and Shannon indices in the CB group compared with the ANT group (p < 0.05), while the Ace index in the CB group was significantly higher than that of the CON group (p < 0.05). Furthermore, by using 16S rRNA gene sequencing, the proportion of Bacteroides in the CB group was increased compared to those in the CON and ANT groups at the genus level. In conclusion, C. butyricum supplemented into feed could improve the growth performance and feed utilization of broilers by promoting immune and intestinal barrier function and benefiting the cecal microflora.