AbstractChiral anomaly, a non-conservation of chiral charge pumped by the topological nontrivial gauge fields, has been predicted to exist in Weyl semimetals. However, until now, the experimental signature of this effect exclusively relies on the observation of negative longitudinal magnetoresistance at low temperatures. Here, we report the field-modulated chiral charge pumping process and valley diffusion in Cd₃As₂. Apart from the conventional negative magnetoresistance, we observe an unusual nonlocal response with negative field dependence up to room temperature, originating from the diffusion of valley polarization. Furthermore, a large magneto-optic Kerr effect generated by parallel electric and magnetic fields is detected. These new experimental approaches provide a quantitative analysis of the chiral anomaly phenomenon which was inaccessible previously. The ability to manipulate the valley polarization in topological semimetal at room temperature opens up a route towards understanding its fundamental properties and utilizing the chiral fermions.