Objective Rheotaxis, namely to orient and swim against the water flow, is a conserved behavior across most fish and amphibians. While the study of rheotaxis behavior has a relatively long history, and in recent years the behavioral algorithm of rheotaxis has been described, how distributed neural circuits integrate multisensory information, make decisions, and generate counterflow motor sequences remain largely unknown. Whole brain calcium imaging of larval zebrafish during rheotaxis would provide a unique opportunity to tackle this difficult problem.Methods To this end, we developed a microfluidic device that can precisely control the water flow and elicit rheotaxis behavior. By integrating the chip with a customized light field tracking microscope, we built a system to record whole brain neural activity in freely behaving larval zebrafish during rheotaxis.Results Larval zebrafish showed reliable rheotaxis behavior in the setup, represented by prominent positional holding and counterflow swimming bouts in water flow. In the meanwhile, we successfully recorded zebrafish whole brain neural activity, from which a few brain regions were identified whose calcium signals strongly correlated with rheotaxis behavior.Conclusion Our study, for the first time, demonstrates a method for imaging whole brain neural activity in larval zebrafish while the animal is performing rheotaxis. Future analysis and modelling of the neural activity and behavioral data will deepen our understanding of sensorimotor transformation in this important naturalistic behavior.