Penicillium rubens, formerly Pencillium chrysogenum, is a filamentous fungus of biotechnological importance because it is one of the main producers of the antibiotic penicillin. However, despite its importance, the regulation of biological processes in this fungus is far from being understood. In fungi, one of the most interesting regulators are zinc finger proteins containing a Zn(II)2Cys6 domain. The genes that encode for these kinds of proteins are found almost exclusively in fungi. The P. rubens genome contains many genes encoding proteins with Zn(II)2Cys6 domains, but only two of them have been studied so far. In this work, we have used CRISPR-Cas9 technology to inactivate the pcz1 gene in P. rubens, which encodes a Zn(II)2Cys6 protein. Inactivation of pcz1 decreases the production of penicillin in P. rubens. In addition, the inactivation of pcz1 decreased growth and conidiation in the fungus but increased conidial germination. Overall, our results suggest that pcz1 would be a positive regulator of penicillin production, growth and conidiation, but it would act as repressor of conidial germination in P. rubens.