Macrophages as professional phagocytes have been considered prominent participants in the response to acute infection and play a significant role in preventing infecting bacteria multiplying and damaging the host environment. Macrophage may also contribute to the inflammatory process by excessive production of reactive oxygen species (ROS) and nitric oxide (NO) which although beneficial in killing bacteria may result in deleterious consequences, especially following an interaction with superoxide radicals to form reactive nitrogen species such as peroxynitrite. Formation of free radicals in macrophages may be regulated by probiotic bacteria (Kumar et. al, 2007). However, the mechanism of regulation is not clear. Thus, studies were carried out to determine the role of probiotic in the acute regulation of both ROS and NO production in the murine J774 macrophages. In this study a cell free Lactobacillus rhamnosus GG culture medium (LGG-CM) was used since it has been shown that LGG releases a number of soluble factors which are responsible for beneficial health effects in the hosts. J774 macrophages were loaded with either H2-DCFDA for monitoring reactive oxygen species or with DAFFM-DA for nitric oxide. Acute free radicals production was measured on a fluorescence microplate reader and changes were analysed by cumulative sum (CuSuM) calculations. The fluorescence measurements were taken every two minutes for the first 60 minutes to monitor free radicals production during ingestion period and from 60 minutes to 280 minutes to monitor free radicals production during digestion period (de-koning ward et al., 1998) by J774 macrophages. The fluorescence was measured at 485 nm excitation and 528 nm emissions. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% LGG-CM and 100% LGG-CM) significantly enhanced ROS generation (p≤.01) but also significantly reduced NO (p≤.05) level in both ingestion and digestion phase of phagocytosis. ROS production in ingestion phase is significantly higher that the digestion phase (p≤.001). These effects of LGG-CM were not altered in the presence or absence of E. coli. A balanced production of NO and ROS is necessary for normal phagocytic function of macrophages. Our findings suggest that probiotics may accelerate bacterial killing by potentially enhancing ROS production and may additionally reduce deleterious effects associated with excessive NO by suppressing production of the latter. In this study a pulse of excessive ROS production to LGG-CM seems to be targeted for rapid digestion of E.coli. Therefore, the ability of probiotic to balance NO and ROS generation can be a novel approach in improving the intestinal homeostasis.