We studied the effects of C-Phycocyanin (C-PC), a biliprotein from Spirulina platensis on the 2-acetylaminofluorene (2-AAF)-induced expression of MDR1, encoded by the multidrug resistance (MDR1) gene, in mouse macrophage cell line (RAW 264.7). Our experimental and In silico studies revealed a significant inhibition of 2-AAF-induced expression of MDR1 protein in C-PC treated mouse macrophage cell line. MDR1 induction by 2-AAF was dependent on ROS (reactive oxygen species)-Akt (protein kinase B)-NF-kappaB (Nuclear factor kappa B) signaling pathway. Generation of ROS, phosphorylation of Akt and corresponding nuclear translocation of NF-kappaB, the events that play a major role in the induction of MDR1 expression, were decreased significantly in C-PC treated cells. NADPH oxidase inhibitor, DPI (Diphenyl iodide), and pharmacological inhibitor of Akt, Akt inhibitor IV, also showed a reduction in MDR1 expression, although not to the same extent as C-PC mediated inhibition of MDR1 expression. To further understand the mechanism, we created a computational model of the detailed ROS-Akt-NF-kappaB pathway. C-PC was modeled purely as a ROS scavenger and this representation matched the experimental trends accurately. Also the ROS levels determined through In silico investigation showed that C-PC was more effective in reduction of MDR1 expression than inhibitors of NADPH oxidase and Akt. Our experimental and In silico studies collectively suggest that 2-AAF induces MDR1 by ROS dependent pathway and C-PC is a potential negative regulator of MDR1 expression. This down regulation of MDR1 expression, induced by xenobiotics such as 2-AAF, suggests C-PC's usefulness in overcoming the drug resistance in cellular systems.