Low plasma CsA concentrations (<300–350 ng/mL) early following allogeneic hematopoietic stem cell transplantation (HSCT) is associated with an increased risk of developing acute graft-versus-host disease (aGvHD). Nevertheless, the current optimal target trough concentration for CsA following HSCT is considered to be 200–400 ng/mL. Here, we performed a retrospective analysis of a homogeneous group of 129 patients who received HSCT after non-myeloablative conditioning, and we analyzed the impact of CsA trough concentration measured during the first four weeks (CsA W1-4) on the incidence aGvHD, relapse-free survival (RFS), non-relapse mortality (NRM), overall survival (OS), and toxicity. The 180-day incidence of grade II-IV aGvHD was 25% (32/129 patients). In multivariate analysis the incidence of grade II-IV aGvHD was significantly lower among patients with a CsA W1-4 concentration 350 ng/mL compared to patients with a concentration <350 ng/ mL (18% versus 38%, respectively; P = 0.007), with a hazard ration (HR) of 0.38 (95% CI: 0.19–0.77). In contrast, we found no correlation between CsA trough concentration and RFS, NRM, or OS. Moreover, we found an increased incidence of hypomagnesemia at higher CsA concentrations, but no difference in the incidence of acute renal toxicity, hepatic toxicity, or electrolyte imbalance. Interestingly, 30% of patients experienced hyponatremia with no apparent cause other than the use of CsA, with urinalysis suggesting SIADH as the underlying cause. Our findings suggest that a CsA trough concentration of 350–500 ng/mL might be more appropriate in the first month following non-myeloablative HSCT.