Bagasse spraying wastewater (BSW) is a source of organic pollutants during bagasse processing. In this study, the feasibility of anaerobic treatment of BSW under different calcium concentrations (60 to 2400 mg/L) was studied. The experiment was performed in a lab-scale up-flow multistage anaerobic reactor (UMAR) inoculated with granular sludge, and operated for 160 days at a constant organic loading rate of 6 kg COD/(m3·d). Treatment of BSW with 60 to 800 mg Ca2+/L resulted in 80.7 to 82.7% of COD removal, 161 to 232.7 mg COD/L of volatile fatty acid (VFA) yield, 0.56 to 0.79 m3/(kgCOD·d) of biogas production rate, and 2.4 to 2.66 m3/(m3·d) of volume loading rate (VLR). The pH remained within the optimal range for anaerobic digestion (adjust to pH = 6.8 to 7.0). The VFAs were composed of 77 to 85% acetic acid, 8.4 to 13.2% butyric acid, and 6.6 to 9.6% propionic acid. At higher influent calcium concentrations (> 800 mg/L), the hydrolysis process appeared to be inhibited, affecting the anaerobic digestion performance of the reactor. In particular, the COD removal efficiency decreased to 55.5%, and the VFA content in the effluent significantly increased due to the lower pH. Microbial community analysis showed that at the end of anaerobic digestion, the Syntrophobacter disappeared, and Clostridium and Anerolineaceae were the main genus and family, respectively. Overall, the results indicated that low calcium (< 300 mg/L) had a positive effect on the UMAR performance.