An experiment on 24 lactating crossbred cows assessed the possibility of utilization of poultry litter (PL) in replacement of conventional protein supplement (soybean meal) in lactating cow diets. Cows of similar age and lactation were randomly divided in four equal groups and fed for 90 days with ad-libitum sorghum fodder and the concentrate mixture was offered to meet energy and protein requirements as per NRC (1988) recommendation. Concentrate fed to control (concentrate I) animals contained de-oiled soybean meal (DSM) as protein supplement while DSM protein was replaced at 25, 37.5 and 50 % with PL in concentrate II, concentrate III and concentrate IV, which accounted 28, 42 and 56 % PL levels respectively, and these concentrate mixtures were fed to cows of T1, T2, T3 and T4 groups respectively. The PL contained OM 71.5, CP 18.0, NDF 55.5, ADF 41.2, Ca 5.4, P 1.7 % and energy 3.75 Mcal/kg DM. The dry matter intake (DMI), milk production and milk composition were similar among four cow group. The DMI varied from 2.54 to 2.72 % of body weight, while milk production ranged from 5.06 to 6.59 kg. Although, milk production and organic matter intake were similar among four groups, however reduction in milk production 6 (T2), 19 (T3) and 22 (T4) % compared to T1 was accompanied by reduced organic matter intake 3, 5 and 9 per cent respectively in cow groups consuming concentrate having PL 28, 42 and 56 percent. PL inclusion in concentrate mixture reduced organic matter and energy contents, while NDF, ADF, Ca and P increased. Digestibility of DM was higher in T1, whereas intake and digestibility of OM, CP, NDF and ADF were similar among four cow groups. Present study concluded that poultry litter can be included in the concentrate mixtures of lactating cows up to 56 percent in replacement of 50 % conventional protein source, without significant reduction in nutrient digestibility and milk production. However, economics of PL inclusion in relation to milk production needs further studies.