Mixed convection flow, heat transfer and entropy generation in a three dimensional rectangular channel under the influence of opposed buoyancy are investigated numerically. Results are obtained for various buoyancy parameters of -500 <= Gr/Re <= 0 at a constant Prandtl number of 0.7 in a symmetrically cooled channel with uniform wall temperature. The flow reversal is found to occur at the four corners between neighboring walls in the entrance region when buoyancy parameter exceeds a certain critical value, which strongly affects the temperature field, local and average Nusselt number, and local and global entropy generation. Such critical buoyancy parameter increases with the increasing aspect ratio of the rectangular cross section for 0.25 <= AR <= 1 and is independent of Reynolds number for 100 <= Re <= 500. The opposed buoyancy force decreases the global entropy generation by heat transfer in the channel, but the entropy generation by fluid friction drastically increases with the increasing opposed buoyancy force. (C) 2015 Elsevier Ltd. All rights reserved.