The immunomodulatory activity of the arabinoxylans (AXs) extracts from cereal sources has been reported to impart health benefits in terms of immune enhancement. This study investigated the effect of enzymatic extraction on extraction yield and structure of AXs from wheat flour pentosan fraction. Under the optimised conditions, the extraction yield of AXs reached up to 81.25%. Furthermore, the study determined whether water-extracted AXs (WEAXs) and enzyme-extracted AXs (E-WEAXs) from wheat flour were able to differentially stimulate nitric oxide (NO) secretion through increased levels of inducible nitric oxide synthase (iNOS) in human U937 monocytes. The results indicated that AXs concomitantly induced (P < 0.05) both NO and iNOS productions in U937 monocytes compared to untreated cells. Compared with WEAXs, E-WEAXs resulted in a higher proportion of low Mw (1–10 KDa) AXs (49.51% vs. 19.11% in WEAXs), a higher A/X ratio (0.83 vs. 0.48 in WEAXs) and a higher yield (12.83 ± 0.35% vs. 7.54 ± 0.47% in WEAXs). Moreover, E-WEAXs induced significantly (P < 0.05) greater NO and iNOS production per million viable cells (61.8 ± 2.7 μm and 42.41 ± 3.83 ng respectively) than WEAXs (51.6 ± 2.6 μm and 33.46 ± 1.48 ng, respectively). The findings suggest AXs may heighten innate immune activity in the absence of infection or disease through an iNOS-mediated stimulation of NO production. The immunomodulatory activity of the wheat-derived AXs was enhanced by enzyme treatment, with low Mw and high A/X ratio associated with elevated NO/iNOS levels in human monocytes compared to water extraction.