Mucin 2 (MUC2) is a critical component of the intestinal mucus barrier. Lactic acid bacteria (LAB) strains can improve mucosal homeostasis. In this study, we determined the expression of Muc2 induced by dead bacteria and cell-free conditioned medium (CM) of 50 LAB strains in the human goblet cell line, LS174T. Dead bacteria or CM of LAB affected the Muc2 expression in a species- and strain-specific manner under homeostasis. Next, LAB strains with different regulatory abilities were selected, gavaged into mice, and exposed to dextran sodium sulfate (DSS) after 1 week. Different LAB strains inhibited intestinal injury to different degrees, with Lactobacillus acidophilus FCQHC4L1 exerting the most potent effect. FCQHC4L1 significantly decreased the secretion of pro-inflammatory factors, promoted the expression and secretion of mucin, and inhibited colitis development. This strain also regulated the gut microbiota and increased the secretion of butyric acid. Moreover, CM of FCQHC4L1 inhibited endoplasmic reticulum (ER) stress and ameliorated the abnormal expression of MUC2 by suppressing the activation of the GRP78/ATF6 and GRP78/IRE1/XBP1 signaling pathways. Our results highlight the potential of FCQHC4L1 as a therapeutic agent for strengthening the mucus barrier and improving the gut health.