In wild-type phototrophic organisms, carotenoids (Crts) are primarily packed into specific pigment-protein complexes along with chlorophylls or bacteriochlorophylls and play important roles in the light reactions of photosynthesis. Diphenylamine (DPA) inhibits carotenogenesis but not phototrophic growth of anoxygenic phototrophs and eliminates virtually all Crts from photosynthetic membranes and photocomplexes. To investigate the effect of Crts on assembly of the reaction center–light-harvesting (RC–LH) complex from the filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii, we generated carotenoidless (abbreviated Crt-less, used here to describe an RC−LH complex whose carotenoid content is greatly reduced but is not zero) RC–LH complexes by growing cells in the presence of DPA. Here we present cryo-electron microscopy structures of the Rfl. castenholzii native and Crt-less RC–LH complexes with resolutions of 2.86 Å and 2.85 Å, respectively. From the high-quality map obtained in this work, several important but previously unresolved details in the Rfl. castenholzii RC–LH structure were determined unambiguously including the assignment and likely function of three small polypeptides, the content and spatial arrangement of Crts with bacteriochlorophyll molecules in the complex. The overall structures of carotenoid-containing and Crt-less complexes are similar. However, structural comparisons showed that only five Crt molecules remain in complexes from DPA-treated cells and that the subunit X (TMx) flanked on the N-terminal helix of the Cyt-subunit is missing. Based on these results, the function of Crts in the assembly of the Rfl. castenholzii RC–LH complex and the molecular mechanism of quinone exchange is discussed. These structural details provide a fresh look at the photosynthetic apparatus of an evolutionary ancient phototroph as well as new insights into the importance of Crts for proper assembly and functioning of the RC–LH complex.