Urban stormwater contains substantial loads of Cu, Pb and Zn, which are considered as key stormwater contaminants. Stormwater biofiltration is a promising option to treat these contaminants. Biofilters are exposed to an alternate cycle of drying and wetting, and the influence of this on pollutant removal performance is as-yet unknown. To investigate the effect of drying and subsequent rewetting on the retention of heavy metals by stormwater biofilters, a laboratory study has been conducted using three groups of biofilter columns, which were dosed with semi-synthetic stormwater according to three different drying and wetting regimes. Some biofilters were fitted with a submerged zone combined with a carbon source, at the bottom of the filter. Overall, the biofilters were very effective in heavy metal removal, provided that they received regular stormwater input. However, after drying extending to three or four weeks, removal of heavy metals decreased significantly. A statistically significant correlation between antecedent dry days and metal removal was shown. Furthermore, a clear effect of the submerged zone was found: after extended dry periods, biofilters with this feature performed significantly better than those without it. In particular, the removal of Cu was clearly increased both during wet and dry periods; for Pb the negative effect of drying was completely eliminated by introducing a submerged zone.