The current study examined the cell adherence property of probiotic Pediococcus pentosaceus GS4 (MTCC12683) with the characterization and functionality in adherence of its surface layer protein (GS4-Slp). The Slp of P. pentosaceus GS4 was extracted purified and detected using SDS-PAGE (98 kDa) and size exclusion chromatography. The cell adherence property of probiotic GS4 (Slp+/Slp−) was evaluated on buccal cells and HCT-116. Purified Slp was found neutralized with raised anti-Slp showing reduced adherence to HCT-116 as evident from SEM analysis. The structure of GS4-Slp was determined by MALDI-TOF analysis, CD analysis, atomic force microscopy (AFM), and FT-IR spectrometry. In Silico approach revealed its indirect similarity with cell membrane protein of Helicobacter pylori. Results thus reveal that GS4 has the potential of the production of 98 kDa Slp which facilitates the cell adherence property. This added probiotic attribute will enhance the probiotic potentials of P. pentosaceus GS4 to use it biotechnologically. Significance: Probiotic Pediococcus pentosaceus GS4 facilitates demonstrable colonization by the elaboration of Slp. This property imparts a value to the strain and claims to be more useful biotechnologically. © 2020 Elsevier B.V.