In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5' UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5' UTR, we injected the GFP reporter mRNA having intact Dsx1α 5' UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.