Microbial reductive dechlorination hosts great promise as an in situ bioremediation strategy for polychlorinated biphenyls (PCBs) contamination. However, the slow dechlorination in sediments limits natural attenuation. Short-chain fatty acids, as preferred carbon sources and electron donors for dechlorinating microorganisms, might stimulate PCB dechlorination. Herein, two sets of short-chain fatty acids, sole acetate and a fatty acid mixture (acetate, propionate, and butyrate), were amended periodically into Taihu Lake (China) sediment microcosms containing nine PCB congeners (PCB5, 12, 64, 71, 105, 114, 149, 153, and 170) after 24 weeks of incubation. Short-chain fatty acids facilitated the long-term PCB dechlorination and the promoting effect of the fatty acid mixture compared favorably with that of sole acetate. By the end of 108 weeks, the total PCB mass concentrations in acetate amended and fatty acid mixture amended microcosms significantly declined by 7.6% and 10.3% compared with non-amended microcosms (P < 0.05), respectively. Short-chain fatty acids selectively favored the removal of flanked meta and single-flanked para chlorines. Notably, a rare ortho dechlorination pathway, PCB25 (24-3-CB) to PCB13 (3-4-CB), was enhanced. Supplementary fatty acids significantly increased reductive dehalogenases (RDase) gene pcbA5 instead of improving the growth of Dehalococcoides. These findings highlight the merits of low cost short-chain fatty acids on in situ biostimulation in treating PCBs contamination. Display omitted •The role of carbon source on long-term dechlorination of PCBs was investigated.•Short-chain fatty acids supplementation facilitated the long-term dechlorination of PCBs•The promoting effect of fatty acid mixture (acetate, propionate, and butyrate) outperformed sole acetate.•The flanked meta and single-flanked para dechlorination were enhanced, as well as a rare ortho dechlorination pathway.•PcbA5 was the main induced RDase gene under the biostimulation of carbon source.