Densification of biomass enables more efficient logistics in a biobased society. When pretreating densified material prior to enzymatic conversion showing positive, neutral, and negative effects of densification have been reported without a clear understanding of the underlying reasons. This study investigates the effects of preheating prior to briquetting of wheat straw on subsequent hydrothermal pretreatment for enzymatic conversion to fermentable sugars. Wheat straw was densified to make briquettes applying; no preheating, preheating at 75°C and 125°C for 5 or 10 min, respectively. Subsequent hydrothermal pretreatment was done for un-briquetted wheat straw and wheat straw briquettes. Enzymatic hydrolysis was performed for all unpretreated and pretreated samples. The results indicated that briquetting itself had no direct effect on enzymatic sugar release from wheat straw (without pretreatment), while preheating at 125 ℃ for 5 or 10 min before briquetting promoted sugar yield. When combined with pretreatment, briquetting showed neutral effect (no preheating or preheating at 75℃ for 5 min), or slight negative effects (preheating at 125℃ for 5 min), or significant negative effects (preheating at 75, 125℃ for 10 min) on sugar yield. Preheating for 10 min at 75 and 125℃ before briquetting may result in irreversible hornification-like effects, which hindered the removing of hemicellulose during pretreatment, and decreased the accessibility of cellulose in enzymatic hydrolysis. Hydrothermal pretreatment played a more important role than briquetting in the structure modification and sugar release of biomass. The hornification-like effects played the main negative role in hydrothermal pretreatment and sugar conversion of biomass.