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A series of batch tests were conducted to elucidate the effect of cultivation temperature on hydrogen production from xylose (20 g COD L−1) using natural anaerobic mixed culture (dominated by Clostridium species). Hydrogen production efficiency was evaluated using the hydrogen yield (HY) and maximum hydrogen production rate (HPRmax). The tested temperature range was from 30 to 55 °C at a 5 °C interval. During fermentation, the hourly variation tendencies of xylose degradation, hydrogen production, ORP and pH were consistent with each other. At the end of fermentation, the hydrogen production, liquid production and biomass concentration variations were temperature dependent. The HY values were in two magnitudes with 1.3 and 0.6–0.8 mol-H2 (mol-xylose)−1 at 30–40 °C (mesophilic) and 45–55 °C (thermophilic), respectively. The HPRmax values were also in two magnitudes with 0.53–1.08 and 0.06–0.20 mol-H2 L−1 d−1 at mesophilic and thermophilic ranges, respectively. These values were 60% of those from an enriched pure culture. Based on having peak HY and HPRmax the optimal cultivation temperature was 40 °C. The liquid metabolite included ethanol, acetate, propionate and butyrate; their concentrations were also temperature dependent. Thermophilic cultivation resulted in high biomass concentration and metabolic pathway shift and then drew to low hydrogen production. Strategies based on cultivation temperature control for optimal hydrogen production from xylose using sewage sludge microflora are proposed.