A systematic study on the interactions between dimethyl sulfoxide (DMSO) and an ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate (EmimAc), during lignocellulosic biomass pretreatment was performed. In this work, the ratio of biomass to EmimAc was fixed at 5 and 10 wt% while different amounts of DMSO were added to EmimAc: 0, 14, 32, 58, 72, 90, 95 and 100 wt%. This enabled a mechanistic understanding of the role of DMSO in IL pretreatment. While enhanced cellulose dissolution upon addition of DMSO was suggested by the XRD data, significantly higher concentrations of DMSO however retarded cellulose dissolution. The effects of DMSO on biomass pretreatment were discussed. Importantly, total sugar conversion of pretreated biomass samples showed a dependence on DMSO concentration, demonstrating the feasibility of using high concentrations of DMSO for efficient biomass pretreatment. This dependence was correlated with cellulose I lattice distortion or transformation during biomass pretreatment. Insights into the interactions between DMSO, EmimAc and biomass were revealed.