Abstract Introduction Mesothelin is expressed in many cancers, especially in mesothelioma and lung, pancreatic and ovarian cancers. In the present study, we evaluate 111In labeled antimesothelin antibodies as an imaging bioprobe for the SPECT imaging of mesothelin-expressing tumors. Methods We radiolabeled the antimesothelin antibodies mAbMB and mAbK1 with 111In using the p-SCN-bn-DTPA chelator. The immunoreactivity, affinity ( K d ) and internalization properties of the resulting two 111In labeled antibodies were evaluated in vitro using mesothelin-expressing A431K5 cells. The biodistribution and microSPECT/CT imaging studies with 111In labeled antibodies were performed in mice bearing both mesothelin positive (A431K5) and mesothelin negative (A431) tumors. Results In vitro studies demonstrated that 111In-mAbMB bound with a higher affinity ( K d =3.6±1.7 nM) to the mesothelin-expressing A431K5 cells than did the 111In-mAbK1 ( K d =29.3±2.3 nM). 111In-mAbMB was also internalized at a greater rate and extent into the A431K5 cells than was the 111In-mAbK1. Biodistribution studies showed that 111In-mAbMB was preferentially localized in A431K5 tumors when compared to A431 tumors. At the low dose, the peak A431K5 tumor uptake of 9.65±2.65% ID/g (injected dose per gram) occurred at 48 h, while at high dose tumor uptake peaked with 14.29±6.18% ID/g at 72 h. Non-specific localization of 111In-mAbMB was mainly observed in spleen. 111In-mAbK1 also showed superior localization in A431K5 tumors than in A431 tumors, but the peak uptake was only 3.04±0.68% ID/g at 24 h. MicroSPECT/CT studies confirmed better visualization of A431K5 tumors with 111In-mAbMB, than with 111In-mAbK1. Conclusion SPECT imaging of mesothelin expressing tumors was demonstrated successfully. Our findings indicate that the antimesothelin antibody mAbMB has the potential to be developed into a diagnostic agent for imaging mesothelin-expressing cancers.