The pluripotency promoting proteins Lin28a and Lin28b act as posttranscriptional repressors of let-7 miRNA biogenesis in undifferentiated embryonic stem cells. The levels of mature let-7a differ substantially in cells lacking Lin28 expression, indicating the existence of additional mechanism(s) of posttranscriptional regulation. Here, we present evidence supporting a role for hnRNP A1 as a negative regulator of let-7a. HnRNP A1 binds the conserved terminal loop of pri-let-7a-1 and inhibits its processing by Drosha. Levels of mature let-7a negatively correlate with hnRNP A1 levels in somatic cell lines. Furthermore, hnRNP A1 depletion increased pri-let-7a-1 processing by cell extracts, whereas its ectopic expression decreased let-7a production in vivo. Finally, hnRNP A1 binding to let-7a interferes with the binding of the KH-type splicing regulatory protein KSRP, known to promote let-7a biogenesis. We propose that hnRNP A1 and KSRP play antagonistic roles in the posttranscriptional regulation of let-7a expression.