One of the drawbacks during second-generation biofuel production from plant lignocellulosic biomass is the accumulation of glucose, the preferred carbon source of microorganisms, which causes the repression of hydrolytic enzyme secretion by industrially relevant filamentous fungi. Glucose sensing, subsequent transport and cellular signalling pathways have been barely elucidated in these organisms. This study therefore characterized the transcriptional response of the filamentous fungus Aspergillus nidulans to the presence of high and low glucose concentrations under continuous chemostat cultivation with the aim to identify novel factors involved in glucose sensing and signalling. Several transcription factor- and transporter-encoding genes were identified as being differentially regulated, including the previously characterized glucose and xylose transporter HxtB. HxtB was confirmed to be a low affinity glucose transporter, localizing to the plasma membrane under low- and high-glucose conditions. Furthermore, HxtB was shown to be involved in conidiation-related processes and may play a role in downstream glucose signalling. A gene predicted to encode the protein kinase PskA was also identified as being important for glucose metabolism. This study identified several proteins with predicted roles in glucose metabolic processes and provides a foundation for further investigation into the response of biotechnologically important filamentous fungi to glucose.