Background: Abnormal high ecotropic viral integration site 1 (EVI1) expression was recognized as a poor prognostic factor in acute myeloid leukemia (AML) patients. However, its prognostic impact in B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains unknown. Procedures: A total of 176 pediatric Ph-negative BCP-ALL patients who received at least 1 course of chemotherapy and received chemotherapy only during follow-up were retrospectively tested EVI1 transcript levels by real-time quantitative PCR at diagnosis and survival analysis was performed. Clinical and EVI1 expression data of 129 pediatric BCP-ALL patients were downloaded from the therapeutically applicable research to generate effective treatments (TARGET) database for validation. Results: In our cohort, the median EVI1 transcript levels were 0.33% (range, 0.0068-136.2%), and 0.10% was determined as the optimal cutoff value for patient grouping by receiver operating characteristic curve. Low EVI1 expression (<0.10%) was significantly related to lower 5-year relapse-free survival (RFS) and overall survival (OS) rates (P=0.017 and 0.018), respectively. Multivariate analysis showed that EVI1<0.10% was an independent adverse prognostic factor for RFS and OS. TARGET data showed that low EVI1 expression tended to be related to a lower 5-year OS rate (P=0.066). Conclusions: Low EVI1 expression at diagnosis may predict poor outcome in pediatric Ph-negative BCP-ALL patients receiving chemotherapy.