Anaerobic wastewater potentially was an ideal medium for cultivating microalgae. The coupled effect of ammonium, temperature and pH on lipids accumulation was a core issue during algal culture using anaerobic wastewater. Therefore, their combined effects on Chlorella pyrenoidosa culture and lipids accumulation in anaerobic effluent were investigated. Free ammonia induced from the rising pH and temperature inhibited algal growth, but significantly promoted lipid accumulation. The highest lipids content reached 30.2% when pH rose to 8.3-8.5 (25 degrees C, ammonium 280 mg/L), which was 1.6-fold higher than that under neutral condition. Moreover, the percentage of unsaturated fatty acids (un-SFAs) increased to 74.8-77.9% at pH 8.3-8.5, whereas it was only 56.1-58.9% under neutral condition. The C18:2 and C18:3 dominated the un-SFAs increase at high pH, typically the percentage of C18:3 increased by 74.5-153.1%. This study provides a potential way for lipid accumulation in algal culture using anaerobic wastewater.