Fisetin (7,3′,4′-flavon-3-ol) is a flavonol found in plants, fruits, and vegetables. It exhibits diverse biological activities, including antioxidant, anti-inflammatory, and anti-cancer effects. However, the low water solubility and bioavailability of fisetin restrict its pharmaceutical applications. In this work, we synthesized a novel fisetin-4′-O-α-D-glucopyranoside (FST-G1) using transglucosylation with sucrose, fisetin, and dextransucrase from Leuconostoc mesenteroides NRRL B-1299CB4. The water solubility of FST-G1 (109.8 ± 6.3 mg/L) was enhanced compared to fisetin (13.6 ± 1.3 mg/L). The antioxidant activities of FST-G1 in non-cellular assays, including ORAC, ABTS•+, and FRAP assays, were lower compared to fisetin. However, FST-G1 exhibited higher nitric oxide inhibition (62.5 µM; 92.3%) in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells compared to fisetin (81.4%). Anti-lipid accumulation in mouse 3T3-L1 cells treated with FST-G1 was similar to that in cells treated with fisetin. Taken together, the novel synthesized FST-G1 is expected to become a promising functional material for using in the pharmaceutical industry.