We previously reported a bi-functional betulinic acid derivative, A12-2 (4), containing an optimized C-28 side chain that exhibits potent anti-HIV activity by inhibiting both HIV-1 entry and maturation. Compound 4 contains C-3 and C-28 side chains that are pharmacophores for anti-HIV maturation and entry activity, respectively. The betulinic acid core, which serves as a molecular scaffold for compound 4, is also important for anti-HIV activity. The main purposes of the present study were to investigate the structure-activity relationships (SAR) of both the C-3 side chain and scaffold of 4. Further modification of the C-3 side chain of 4 suggested that both bulkier and smaller C-3 substituents negatively impacted the anti-HIV-1 activity. SAR study of the scaffold indicated that the betulinic acid moiety of 4 could be replaced with other scaffolds while still remaining active against HIV-1 replication. Among the synthesized compounds, the most effective molecular scaffold for anti-HIV activity remained to be betulinic acid (0.0026 μM), followed by moronic acid, ursolic acid, and oleanolic acid. On the other hand, substitution of the betulinic acid moiety of 4 with glycyrrhetinic acid or lithocholic acid completely abolished anti-HIV activity. Mechanism of action studies indicated that all active terpenoid analogs of 4 retained both anti-HIV-1 entry and anti-HIV-1 maturation activities.