Stillage is an abundant wastewater from the sugarcane ethanol industry. It is rich in fermentable substrates and presents low-nutrient content, constituting a promising substrate for polyhydroxyalkanoate (PHA) production by mixed microbial cultures (MMC). This work assessed the enrichment of a PHA-accumulating MMC from acidified sugarcane stillage in a sequencing batch reactor under increasing organic loading rates (OLR) and no external nutrient supplementation. The OLR was increased from 1.0 to 7.1 kg COD m(-3) day(-1) in four steps. A PHA-producing MMC with high storage response was selected in all experimental conditions. The volumetric biomass productivity and the maximal PHA storage capacity increased continuously with the OLR, reaching 0.061 g VSS L-1 h(-1) and 0.49 g PHA g VSS-1, respectively. The highest observed PHA storage yield (0.60 g CODPHA g COD.t(-1)) and specific PHA storage rate (0.169 g CODPHA g of CODX h(-1)) were obtained for the OLR of 4.5 kg COD m(-3) day(-1). The PHA produced was a co-polymer of 3-hydroxybutyrate (86-77%mol) and 3-hydroxyvalerate (14-23%mol). The performance of the biomass enrichment was comparable to those attained with other agro-industrial wastewaters, indicating the potential of acidified sugarcane stillage as a feedstock for MMC PHA production.