Abstract Background Optimal ustekinumab levels (UST) in Crohn disease (CD) treatment have not been defined. We set out to define the optimal UST to differentiate between remission and active CD, as defined using the serum-based endoscopic healing index (EHI). Methods Paired serum UST and EHI tests were analyzed. Remission was defined as EHI <20. Active disease was defined as EHI ≥50. The proportion of patients in remission was compared across UST quartiles. UST in subjects with EHI <20 and EHI ≥50 were compared. An area under receiver operating characteristic curve was generated to identify an optimal UST to differentiate between active disease and remission. Results A total of 337 unique patients were identified; median UST and EHI were 5.0 µg/mL [interquartile range (IQR) 2.7–9.1] and 37 (IQR 26–53), respectively. EHI <20 (remission) was found in 57 (16.9%) patients. EHI ≥50 (active disease) was found in 97 (28.8%) patients. Higher proportions of subjects were in remission for increasing UST quartiles, P = 0.01. Median UST in patients with EHI <20 and EHI ≥50 were 7.5 µg/mL (IQR 4.6–10.9) and 3.1 µg/mL (IQR 1.8–6.6), respectively, P < 0.001. An UST threshold of 3.75 µg/mL optimally differentiated between active disease and remission (area under the curve 0.725). UST levels >3.75 µg/mL were associated with a lower proportion of subjects with active disease (EHI ≥50; 18.9%) compared with UST levels ≤3.75 µg/mL (45.6%, P < 0.001). Conclusions Using the EHI, we identified a threshold UST level of 3.75 µg/mL to optimally differentiate between active and quiescent CD. These data suggest that UST serum concentrations of >3.75 µg/mL are optimally associated with endoscopic remission in CD.