The objective of this study was to investigate the effects of storing temperature on dissipation of ceftiofur and antibiotic-resistance genes in dairy manure slurry. Feces and urine were totally collected from 5 lactating dairy cows with ceftiofur administration, and then mixed completely to achieve a homogeneous manure status. Manure slurry was generated by mixing manure and sterile water at 1:1. A 200 mL aliquot of slurry was transferred into 400 mL glass beakers and incubated at 10, 25, 35, or 55°C. Each temperature treatment had 4 replicates. The beakers were weighted daily, and sterilized water was added to compensate the weight loss due to water evaporation. Six g of slurry was sampled from each beaker on d 0, 1, 3, 7, 14, 28, 56, and 90 for ceftiofur concentration analysis. Slurry samples from d 0, 3, 7, 28 were used to determine gene expressions of 16S rRNA, CfxA, and TetW using the q-PCR. The q-PCR results were normalized using natural log-transformation. The temperature and incubation time effects were evaluated using a mixed statistical model which included temperature and time as fixed effects with glass beakers as repeated measurements. Significant temperature and time effects were observed for ceftiofur concentrations (P < 0.0001). With increased incubation temperature, the dissipation rate of ceftiofur was increased. Ceftiofur concentration was completed reduced at 55°C on d 14, while 76.2, 70.8, and 84.6% of ceftiofur was removed at 10, 25, and 35°C on d 90. The lowest relative abundance of CfxA and TetW were observed at 55°C on d 28, which reduced around 60% for CfxA and 50% for TetW relative to the initial abundance (P < 0.05). In conclusion, the thermophilic treatment is recommended for the dissipation of ceftiofur and antibiotic-resistance genes in dairy manure slurry.