Although carbon and oxygen isotopic compositions of carbonates are valuable for Quaternary climate reconstruction due to their sensitivity to paleoclimate changes, a comprehensive understanding regarding the formation, factors influencing isotopic variations, and age uncertainties of secondary carbonates in loess deposits remains elusive. This study investigates the microstructure and stable isotopes of secondary carbonates, specifically calcified root cells (CRC) and needle-fiber calcite (NFC), found within the loess deposits of the last glacial period in the northwestern region of the Chinese Loess Plateau. The average δ13C values of soil total organic matter, CRC, NFC, and total inorganic carbonates in the YuanBao sections are −22.1‰, −14.9‰, −7.0‰, −4.6‰, respectively. Radiocarbon dating results reveal that both CRC and NFC were not of synsedimentary origin. The pronounced negativity of the carbon isotopic composition and the microstructures offer insights that CRC formation is closely tied to C3 plants. The synchronous depletion of δ13CCRC and δ18OCRC values during the transition period from the last glacial to the Holocene, underscores the sensitivity of isotopic composition of CRC to local environmental changes, making it a potential candidate for paleo-climate studies. Additionally, CRC formed during the glacial period provides a close approximation to the true sedimentation age. The radiocarbon dating of NFC highlights a significant fluctuation in carbon source translocation depth, resulting in a pronounced temporal and spatial decoupling between NFC formation and the surrounding deposits. Consequently, values of δ13CNFC and δ18ONFC seem to exhibit no significant trend across the limited depth range in this research. We propose that, when using NFC for paleoclimate reconstruction, careful consideration of variations in translocation depths of carbon sources is imperative to prevent misestimations. NFC could potentially be more fitting for broader temporal-scale paleoclimate reconstructions rather than high-resolution investigations. •Loess carbonate NFC exhibiting substantial variations in the depth of carbon source translocation.•CRC formed during loess development could provide a close approximation to sedimentation age.•The isotopic compositions of CRC are responsive to local environmental changes.•Carbonates formed with different mechanisms show distinct isotopic compositions, reflecting various climatic information.