This research aimed to evaluate the effects of Capsicum oleoresin (CAP) supplementation on rumen fermentation in vivo and In vitro, and lactation performance in buffaloes. In the experiment in vitro, 2 × 5 factorial design was carried out according to two temperatures (normal temperature: 39 °C; hyperthermal temperature: 42 °C) and five CAP concentrations (0 mg/L; 2 mg/L; 20 mg/L; 200 mg/L; 2000 mg/L). In the experiment in vivo, four multiparous mid-lactating Mediterranean buffaloes (body weight: 640.08 ± 17.90 kg) were randomly allocated to four treatments according to 4 × 4 Latin square design for CAP supplementation in four dosages (0 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of dry matter). The experiment’s results In vitro showed that hyperthermal temperature affected all fermentation characteristics measured in this research. CAP decreased the pH, short-chain fatty acids concentration, and percentages of propionate, butyrate, isobutyrate, valerate, and caproate, while increasing the percentage of acetate and the ratio of acetate to propionate at normal temperature (p ≤ 0.05). In the experiment in vivo, CAP decreased the percentage of propionate and quadratically affected acetate percentage in rumen fluid (p ≤ 0.05). CAP reduced rectal temperature and respiratory rates (p ≤ 0.05) and tended to increase dry matter intake quadratically (p ≤ 0.10). For lactation performance, CAP increased milk yield and milk lactose yield (p ≤ 0.05), and tended to increase milk protein yield (p ≤ 0.10). In conclusion, CAP modified rumen fermentation characteristics in vivo and In vitro and had beneficial effects on lactation performance in buffaloes during summer.