Antibiotic fermentation residues (AFRs) have been classified as hazardous waste in China. Anaerobic fermentation may be a good approach for AFRs treatment, through which value-added chemicals could be obtained simultaneously. This study firstly explored medium-chain fatty acids (MCFAs) production from AFRs through two-stage anaerobic fermentation, and gamma radiation was adopted for AFRs pretreatment. The results showed that both antibiotics removal and MCFAs production from AFRs were significantly promoted by gamma radiation pretreatment. No residual Cephalosporin C (CEP-C) was detected in gamma radiation treated groups after fermentation. Highest MCFAs concentration of 90.55 mmol C/L was obtained in 50 kGy treated group, which was 2.22 times of the control group. Genera that were positively correlated with MCFAs production were enriched in gamma radiation treated groups, like genus Paraclostridium, Terrisporobacter, Caproiciproducens and Sporanaerobacter, while genera that were negatively correlated with MCFAs production were diminished during the chain elongation process, like genus Bacteroides and NK4A214_group. Enzymes analysis suggested that the promoted MCFAs production was induced by the enrichment of functional enzymes involved in Acetyl-CoA formation and RBO pathway. This work suggested that gamma radiation pretreatment and two-stage anaerobic fermentation could achieve the dual benefits of AFRs treatment and value-added chemicals recovery.