Advances in bone tissue engineering depend on biomaterials that can promote osteogenic differentiation and matrix mineralization for functional bone regeneration. Gellan gum (GG), a naturally derived polysaccharide, which is biocompatible, has tunable gelation, and has potential bioactivity, is considered an ideal candidate for composite scaffolds. This study evaluates the osteogenic potential of two GG variants, low acyl gellan gum (LA-GAGR) and Mini-GAGR, and compares their effects with other polysaccharides, hyaluronic acid (HA), welan gum (WG), and dextran. MC3T3-E1 mouse pre-osteoblast cells were cultured with each polysaccharide in both liquid and hydrogel formulations over 3, 7, and 14 days. Osteogenic responses were assessed through Alizarin Red Staining (ARS) for calcium deposition, alkaline phosphatase (ALP) staining, and gene expression of key osteogenic markers. LA-GAGR (500 kDa) and Mini-GAGR (760 Da) in their hydrogel form showed higher calcium deposition, and upregulation of osteogenesis-associated genes (RUNX2, SP7) and extracellular matrix (ECM)–associated genes (OCN, OPN, and COL1), when compared with other polysaccharides. Notably, Mini-GAGR showed higher effects than LA-GAGR on osteogenic markers, matrix-associated gene expression, while LA-GAGR, with its higher molecular weight, showed higher ALP staining intensity. Overall, these results suggest that LA-GAGR and Mini-GAGR exhibit osteogenic potential under the tested in vitro conditions, supporting their further investigation as polysaccharide-based materials for bone tissue engineering applications.