Colchicine (COL) is a permeability-glycoprotein (P-gp) substrate drug used for familial Mediterranean fever, acute pericarditis, and the management of acute gout. It has a narrow therapeutic index which implies that a small change in the drug's absorption profile may lead to either toxicity or therapeutic failure. Absorption can be altered by modulating the function of P-gp via the concomitant use of drugs, herbal medicines, or food supplements such as probiotics. Here, we investigated the effect of probiotic Lactobacillus acidophilus BIOTECH 1900 on COL's transepithelial mucosal-to-serosal transport in the jejunum of ICR mice. A high-performance liquid chromatography-photodiode array (HPLC-PDA) method for the assay of COL was developed and validated. The HPLC-PDA method was applied in an ex vivo non-everted gut sac model to measure COL's cumulative mucosal-to-serosal transport and apparent permeability (Papp). Treatment of L. acidophilus BIOTECH 1900 resulted to a significantly lower COL transport and Papp value compared to the control group. Additionally, the activity of L. acidophilus BIOTECH 1900 was found to be similar to dexamethasone, a known P-gp inducer. We report that L. acidophilus BIOTECH 1900 decreases the transepithelial mucosal-to-serosal transport of COL, suggesting possible P-gp induction. Further studies are recommended to substantiate this transporter-based drug-probiotic interaction.