Various efforts are currently underway to develop therapeutic agents to arrest the replication of the human immunodeficiency virus (HIV). Although several nucleoside HIV-1 reverse transcriptase (RT) inhibitors (AZT, ddI, ddC, D4T) are approved by the FDA and used clinically, adverse side effects and the development of drug resistant virus have been reported, so compounds possessing potent anti-HIV activity with novel modes of action are urgently needed. Our laboratory focuses on discovering plant-derived natural products as potential new lead compounds for anti-HIV agents and modifying these leads to enhance activity. Previously, we isolated betulinic acid (1) and platanic acid (11) as anti-HIV principles from Syzigium claviflorum; betulinic acid exhibited inhibitory activity against HIV-1 replication in H9 lymphocyte cells with an EC50 value of 1.4 µM and a therapeutic index (TI) of 9.3, while platanic acid had an EC50 of 6.5 µM and TI of 14. Hydrogenation of betulinic acid yielded dihydrobetulinic acid (6), which showed slightly more potent activity (EC50 0.9 µM, TI 14). On this basis, we modified the C3 hydroxy groups of betulinic acid and dihydrobetulinic acid by introducing acyl groups: treatment with 3,3-dimethylglutaric anhydride or diglycolic anhydride gave corresponding 3-O-acyl derivatives (4, 5, 9, 10), while reaction with dimethylsuccinic anhydride afforded isomeric mixtures (2/3, 7/8) separated by preparative HPLC. Anti-HIV assays indicated that 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (3) and -dihydrobetulinic acid (8) demonstrated extremely potent activity in acutely infected H9 lymphocytes with EC50 values of <3.5×10⁻⁴ µM and TI values of >20000 and >14000, respectively. Compounds 4, 5, 9, and 10 also exhibited potent activity (EC50 0.01–2.3×10⁻³ µM, TI 1017–2344). Mechanism studies showed tested compounds did not inhibit HIV-1 RT activity, but some inhibited HIV-induced membrane fusion; however, the 7700-fold higher anti-HIV activity of 3 compared to its isomer 2 (which inhibited syncytia formation at the same concentration) suggests additional mechanisms. This paper describes the preparation of these betulinic acid and dihydrobetulinic acid derivatives and evaluation of their anti-HIV activities.