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<i>Penicillium rubens</i> is a filamentous fungus of great biotechnological importance due to its role as an industrial producer of the antibiotic penicillin. However, despite its significance, our understanding of the regulatory mechanisms governing biological processes in this fungus is still limited. In fungi, zinc finger proteins containing a Zn(II)₂Cys₆ domain are particularly interesting regulators. Although the <i>P. rubens</i> genome harbors many genes encoding proteins with this domain, only two of them have been investigated thus far. In this study, we employed CRISPR-Cas9 technology to disrupt the <i>pcz1</i> gene, which encodes a Zn(II)₂Cys₆ protein in <i>P. rubens</i>. The disruption of <i>pcz1</i> resulted in a decrease in the production of penicillin in <i>P. rubens</i>. This decrease in penicillin production was accompanied by the downregulation of the expression of <i>pcbAB</i>, <i>pcbC</i> and <i>penDE</i> genes, which form the biosynthetic gene cluster responsible for penicillin production. Moreover, the disruption of <i>pcz1</i> also impacts on asexual development, leading to decreased growth and conidiation, as well as enhanced conidial germination. Collectively, our results indicate that <i>pcz1</i> acts as a positive regulator of penicillin production, growth, and conidiation, while functioning as a negative regulator of conidial germination in <i>P. rubens</i>. To the best of our knowledge, this is the first report involving a gene encoding a Zn(II)₂Cys₆ protein in the regulation of penicillin biosynthesis in <i>P. rubens</i>.