<P><B>Abstract</B></P> <P>A pretreatment process using alkali/urea solution at low temperature was proposed for enhanced cellulosic biohydrogen production. Different alkaline solutions with both presence and absence of urea were studied. It can be found NaOH/Urea pretreatment exhibited excellent pretreatment performance at temperature from −8 °C to −20 °C. Microscopic structure observation combined FTIR analysis further demonstrated that NaOH/Urea pretreatment at low temperature could effectively disrupt the structure of rice straw and made more cellulose and hemicellulose available. The pretreated materials were then subjected for biohydrogen production by <I>Thermoanaerobacterium thermosaccharolyticum</I> M18. The maximum hydrogen production and energy conversion efficiency of 22.08 mmol/L and 9.76% were obtained from NaOH/Urea pretreated rice straw at low temperature. The results were 161.92% and 56.91% higher than the counterpart without pretreatment, respectively. This study provides a new direction to pretreat lignocellulose efficiently for enhanced biohydrogen production at cold climate region.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Cold alkali/urea pretreatment was employed to enhance H<SUB>2</SUB> production from rice straw. </LI> <LI> Chemical compositions analysis revealed the efficiency of alkali/urea pretreatment. </LI> <LI> Cold NaOH/Urea pretreatment enabled a high energy conversion efficiency. </LI> <LI> This study provides a new promising way for enhanced H<SUB>2</SUB> production from rice straw. </LI> </UL> </P>