The well-studied xylanase inhibitor protein (XIP-I) regularly inhibits fungi-derived xylanases, yet some fungal xylanases are not inhibited by XIP-I. Based on the sequence alignment with the known XIP-I resistant NpXyn11A from Neocallimastix patriciarum, a new annotated xylanase from ruminal fungus Neocallimastix sp. GMLF1 was found, noted as Xyn1B, which shared 77.8% of sequence identity with NpXyn11A and was proved to be resistant to XIP-I. To evaluate the performance of a XIP resistant xylanase used in barley malt saccharification, a XIP-I sensitive xylanase ThXyn1 from Trichoderma harzianum was chosen as a control. The barley malt saccharification experiment was carried out on the condition with or without extra XIP-I added. The results showed that Xyn1B displayed only slight difference in presence and absence of added XIP-I, with the difference of xylose released (DX) and the difference of mash clarity (DA) being 0.17 g L À1 (P > 0.05) and 0.007 (P > 0.05), respectively; while those for ThXyn1 group reached 0.96 g L À1 (P < 0.01) and 0.095 (P < 0.05), indicating that XIP-I did not adversely affect Xyn1B's function, but did affect ThXyn1's function. Our work for the first time suggested that a xylanase with resistance to xylanase inhibitor proteins might have an advantage in barley malt saccharification over an inhibitor sensitive xylanase.