The objective of this study was to evaluate whether wheat factory sewage (WFS) could partially replace barley grain in the diet of dairy cows without adversely affecting dry matter intake (DMI), ruminal fermentation, digestibility and milk production of dairy cows. Eight multiparous (60 ± 3 days-in-milk) Holstein dairy cows were used in a replicated 4 × 4 Latin square design experiment with four 21-d periods. The four diets (treatment) differed by partially substituting WFS for barley grain at the rate of 0% (WFS0), 4% (WFS4), 6% (WFS6) or 8% of dietary dry matter (DM; WFS8). DM content of diets decreased from 65%, 59%, and 57% to 54% by increasing the inclusion of WFS due to high water content of WFS (80%). DMI changed quadratically and tended (P = 0.08) to be higher for WFS4 (23.1 kg/d) than for other three diets which were similar (21.5 kg/d). Ruminal fluid pH linearly increased (P = 0.01) with increasing WFS in the diet. Ruminal concentrations of total volatile fatty acid (VFA; 100–103 mM), acetate (65–67 mM), propionate (24–25 mM) and ratio of acetate:propionate (2.68:2.89) were not affected by increasing the replacement of barley grain with WFS. Apparent total tract digestibilities of DM (67%), crude protein (CP; 68%) and neutral detergent fibre (NDF; 54%) were not different among treatments. Milk yield (averaged 40 kg/d) and milk composition were not affected by the dietary treatments. These results showed that the inclusion of WFS at rates of 0, 4, 6, and 8% in place of barley grain in dairy cow diets decreased DM content of diets, but had no adverse effects on DMI, rumen fermentation, digestibility and milk production responses. In conclusion, WFS can be a candidate by-product as an alternative feedstuff for the partial replacement of barley grain in lactating dairy cow diets and also considered as an alternative energy source in a cost-effective manner when the price is competitive.