Five novel 16-membered macrolides (1b–3b), homologues of rhizoxin, were isolated from the fungus Rhizopus chinensis, and their structures were determined using physicochemical properties (optical rotation, UV absorption) and spectral data (¹H-NMR, high-resolution electron impact mass spectrometry, HREIMS). Rhizoxin (1a), a previously identified 16-membered macrolide toxin from R. chinensis (the causal agent of rice seedling blight) with potent antifungal and anti-tumor activities, was used as a reference. The R. chinensis Rh-2 strain was cultured, and compounds were purified via solvent extraction (acetone, ethyl acetate), column chromatography (silica gel, LH-20), and HPLC. Structural analysis revealed: 1) The b-series compounds (1b, 2b, 3b) contain one fewer CH₂ unit than corresponding a-series compounds (1a, 2a, 3a), with the 17-OCH₃ group in the a-series replaced by an OH group (evidenced by absent OCH₃ signals and shifted H-17 peaks in ¹H-NMR); 2) Compounds 2a (and 2b) and 3a (and 3b) have one and two fewer oxygen atoms than 1a, respectively, with the C-2,3 epoxy group in 1a replaced by an α,β-unsaturated ester moiety (E-configuration confirmed by large coupling constants of H-2 and H-3); 3) Compound 3a (and 3b) features a double bond at C-11,12 (supported by chemical shift changes of H-11/H-12a and UV spectra indicating a conjugated diene system). These compounds are considered biogenetic precursors of rhizoxin. Biological activity assays will provide insights into the structure-activity relationships of rhizoxinoid compounds.