This study aimed to evaluate the survivability of Lactiplantibacillus plantarum PMO08 in the human gastrointestinal tract and its adaptability in the colon using in vitro models. After exposure to gastric and small intestinal conditions, the majority (92.70 ± 1.14%) of PMO08 was found to be damaged, as determined by confocal microscopy and flow cytometry. During in vitro colonic fermentation, PMO08 not only increased abundance up to 0.47 ± 0.04% compared with the control sample (0.00 ± 0.00%) at 24 h but also facilitated the growth of beneficial or commensal bacteria, thereby increasing the α-diversity indices. Additionally, PMO08 significantly elevated the levels of short-chain fatty acids (SCFAs) and various organic acids. Our results demonstrate that PMO08 possesses moderate viability under gastrointestinal conditions but exhibits superior probiotic activity in the colon.