Although de novo DSA are associated with inferior graft survival, there are no effective strategies to prevent their formation. Underexposure to MPA (prodrug: MMF) also contributes to rejection rates early after transplantation, but the effect of this phenomenon on the formation of DSA long-term post-transplantation is unknown. Data are expressed as mean (standard deviation). All available data from 32 renal transplant recipients (age at transplantation 7.5 [4.5] yr) on tacrolimus and MPA immunosuppression with an average follow-up of 9.4 (s.d. 4.6) yr were analyzed. DSA were measured using the Luminex assay (>500 MFI was considered DSA-positive). Tacrolimus and MPA levels were measured with the Abbot Tacro II and EMIT assay, respectively. Among 1964 MPA and 3462 tacrolimus trough levels, the average MPA trough level was 3.2 (1.5) mg/L and the average tacrolimus level was 6.7 (2.8) ng/mL. At last follow-up, only 5/32 patients had undetectable DSA, with 5/32 having no class I antibodies and 6/32 having no class II antibodies. DSA formation was associated with a lower minimum MPA trough level (0.27 [0.23] vs. 0.47 [0.18] mg) and cystatin C eGFR (48  vs. 70  mL/min/1.73 m2) for class I DSA formers. The average eGFR of patients without class I DSA was 70 (23) mL/min/1.73 m2, whereas the average eGFR of patients with class I DSA was 48 (21) mL/min/1.73 m2 (p = 0.0071). MPA trough levels <1.3 mg/L long-term post-transplantation are associated with the formation of DSA. The association between the formation of DSA and minimum MPA exposure may support a strategy for preventing the formation of DSA.