As a disease caused by an impaired intestinal epithelial barrier, imbalanced flora, immune imbalance and genetic susceptibility, ulcerative colitis (UC) is becoming a health threat for all ages. Lactobacillus acidophilus (L. acidophilus), an attracting probiotic, has already been confirmed to improve immune dysfunction, stabilize intestinal microflora, and combat gut disorders. However, no studies have focused on the effects of different forms of L. acidophilus on UC, and its mechanism involved in the mitophagy/NLRP3 inflammasome pathway has not been reported. In this study, we found that compared with the heat-killed L. acidophilus and the culture supernatant of L. acidophilus, the live L. acidophilus (La) has the optimal therapeutic effect on UC rats. Furthermore, La evidently increased the contents of SCFAs, inhibited NLRP3 inflammasome and facilitated autophagy. SCFAs regulated by La balanced inflammation homeostasis and improved intestinal barrier dysfunctions in vitro and in vivo, which was achieved by activating the mitophagy/NLRP3 inflammasome pathway. Moreover, PCR analysis indicated that the aforementioned effects of SCFAs regulated by La may be due to the activation of G protein-coupled receptors. These findings provided guidance for the application of L. acidophilus in daily life and provided a new molecular target for interactions among L. acidophilus, its metabolites and host immunity.