The importance of intestinal alkaline phosphatase (IAP) in maintaining gut health and intestinal homeostasis is well established. The objective was to investigate the tolerance of poultry and swine to dietary supplementation of a novel microbial-derived alkaline phosphatase (AP; E.C. 3.1.3.1 produced by Paenibacillus lentus strain CMG3709). Studies were conducted on day-old Ross 308 chicken (n=1,000; Study 1) and weaned piglets (n=180; Study 2) for a duration of 42 days; and consisted of four treatment groups (TG) based on the concentration of microbial-derived AP supplemented in their diet at 0; 12,000; 20,000; and 200,000 U/kg of feed. Parameters such as animal survival, hematology, coagulation, and biochemical indices were assessed at the end of the study. The effect of microbial AP on nutrient absorption through skin pigmentation and intestinal permeability were also investigated in broilers (n=600; Study 3). In poultry (Study 1), there were no statistically significant differences between control and TG for any of the hematological and biochemical parameters, except for a marginal increase (P < 0.05) in serum phosphorus at the highest dose. This variation was not dose-dependent, was well within the reference range, and was not associated with any clinical correlates. In swine (Study 2), hematological parameters such as leukocyte, basophil, and lymphocyte counts were lower (P < 0.05) for the two highest doses but were traced back to individual variations within the group. The biochemical indices in piglets showed no significant differences between control and supplemental groups except for glucose (P = 0.0005), which showed a high effect (P = 0.008) of the random blood collection order. Nonetheless, glucose was within the normal reference range, and were not related to in-feed supplementation of AP as they had no biological significance. The survival rate in all three studies was over 98%. Dietary supplementation of microbial-derived AP up to 16.7 times the intended use (12,000 U/kg feed) level had no negative effects in both poultry and swine. Infeed supplementation of microbial-derived AP for 28 days improved intestinal pigment absorption (P < 0.0001) and reduced intestinal paracellular permeability (P = 0.0001) in broilers (Study 3). Based on these results, it can be concluded that oral supplementation of microbial-derived AP is safe for poultry and swine and effective at improving gut health in poultry.