Diacetylated lactonic sophorolipids (polyLSL[6'Ac,6?Ac]), a biosurfactant, can be efficiently polymerized by ring-opening metathesis polymerization (ROMP). In this paper, enzyme-mediated chemical transformations are developed to regioselectively modify LSL[6'Ac,6?Ac] at sophorose primary hydroxyl positions (6' and 6?). The resulting modified LSLs were polymerized to expand polyLSL structural diversity, that is, polyLSL[6'OH,6?Ac], polyLSL[6'OH,6?OH], polyLSL[6'Bu,6?Ac], polyLSL[6'N3,6?Ac], and polyLSL[6'MA,6?Ac]. Controlled placement of azide and methacrylate at sophorolipid moieties enables the use of "click" reactions to introduce bioactive groups. Thermal analyses of polyLSLs showed that the acylation pattern at sugar moieties has a remarkable effect on chain stiffness and crystallinity. Films of polyLSL[6'Ac,6?Ac], polyLSL[6'OH,6?Ac], and polyLSL[6'Bu,6?Ac] exhibited nonbrittle behaviors with compressive elastic moduli ranging from similar to 1.5 to similar to 4.9 MPa. PolyLSLs were cytocompatible with human mesenchymal stem cells (h-MSCs), and examination of material-induced osteogenic cell lineage progression uncovered a dependence on polyLSL substitution at sophorose 6'-sites. This research reveals opportunities to regulate polyLSL physical properties and cell response behaviors by variation in substituents at polyLSL sophorolipid moieties.