Papuamides A (1a) and B (1b) are novel cyclic depsipeptides isolated from Papua New Guinea collections of the marine sponges Theonella mirabilis and Theonella swinhoei, exhibiting a strong inhibitory effect on the infection of human T-lymphoblastoid cells by HIV-1RF in vitro with an EC50 value of about 4 ng/mL. Recent intensive efforts have been directed towards gaining synthetic access to these natural products and establishing the stereochemistry of some sterogenic centers within this class of compounds, but none of these molecules have yet been assembled. Herein, we report our synthetic strategy towards papuamide B. A convergent route to natural papuamide B has been developed, which features stereoselective assembly of its dienoic acid fragment and an efficient elaboration of the (2S,3S,4R)-3,4-dimethylglutamine residue through a 3,4,5-trisubstituted δ-valerolactone. Accompanying the total synthesis, the configuration of three sterogenic centers of its 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid unit was established, and the stereochemistry of its 2,3-diaminobutanoic acid segment was revised from the originally assigned 2S,3R to 2S,3S. This approach will be of great benefit for the total synthesis of other anti-HIV cyclic depsipeptides such as callipeltin A and neamphamide A, aids in fully establishing the structures of related cyclodepsipeptides like papuamide A and mirabamides A–D, and opens the door for elaboration of related natural products and their analogues, which would prompt the structure–activity relationship (SAR) studies of these potent anti-HIV agents.