Spent biomass from agricultural and forestry industries are substantial low-cost carbon source for reducing the input of microbial lipid production. Herein, the components of the vineyard winter prunings (VWPs) from 40 grape cultivars were analyzed. The VWPs contained (w/w) cellulose ranged from 24.8% to 32.4%, hemicellulose 9.6% to 13.8%, lignin 23.7% to 32.4%. The VWPs from Cabernet Sauvignon was processed with the alkali-methanol pretreatment, and 95.8% of the sugars was released from the regenerated VWPs after enzymatic hydrolysis. The hydrolysates from the regenerated VWPs was suitable for lipid production without further treatment as a lipid content of 59% could be achieved with Cryptococcus curvatus. The regenerated VWPs was also used for lipid production via simultaneous saccharification and fermentation (SSF), which led to a lipid yield of 0.088 g/g raw VWPs, 0.126 g/g regenerated VWPs and 0.185 g/g from the reducing sugars. This work demonstrated that the VWPs can be explored for co-production of microbial lipids.