Abstract Purpose Stroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB₂R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [¹⁸F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI). Procedures Our recently developed CB₂R PET tracer [¹⁸F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB₂R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [¹⁸F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T₂-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice. Results mRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3–2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [¹⁸F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [¹⁸F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [¹⁸F]RoSMA-18-d6 was confirmed only in the CB₂R-rich spleen. Conclusions This study revealed an increased [¹⁸F]RoSMA-18-d6 measure of CB₂R and a reduced [¹⁸F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [¹⁸F]RoSMA-18-d6 might be a promising PET tracer for detecting CB₂R alterations in animal models of neuroinflammation without neuronal loss.