Safety assessment of graphene-based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here we compared the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice. Micrometre-sized GO induced stronger pulmonary inflammation than nanometre-sized GO, despite a reduced translocation to the lungs. Genome-wide RNA sequencing also revealed distinct size-dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggered the formation of granulomas that persisted for up to 90 days, no pulmonary fibrosis was observed. These latter results could be partly explained by the progressive biotransformation of GO sheets into less graphitic structures, as evidenced by Raman imaging. Our findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometre-sized GO should be avoided in production plant or applications, where aerosolised dispersions are likely to occur. These results are important towards the implementation of a safer-by-design approach for GBM enhanced products and applications, for the benefit of workers and end users.