Ampicillin has been widely used to treat bacterial infections. When we used ampicillin to eliminate bacterial contamination in yeast cultures, we observed induction of phosphorylation of MAP kinase 1 (Mpk1), a previously unknown function of ampicillin. We therefore investigated whether ampicillin activates the signal transduction pathway. Phosphorylation of Saccharomyces cerevisiae Mpk1 was induced by ampicillin in a dose- and time-dependent manner through the PKC1-CWI pathway. Mpk1 phosphorylation was maximal after treatment with 3 mM ampicillin for 90 min. Despite activation of Mpk1 phosphorylation, ampicillin did not influence yeast cell growth. Ampicillin reduced miconazole antifungal activity; miconazole had a minimum inhibitory concentration of 3.125 µg/mL against Candida albicans, which increased to 25 µg/mL after 48 h of treatment with 3 mM ampicillin. Finally, ampicillin activated phosphorylation of ERK1/2 (a mammalian homolog of Mpk1), with maximum effect at 3 mM ampicillin, in human HepG2 cells, but did not influence cell viability. The results of this study clearly indicate that ampicillin activated Mpk1 phosphorylation in yeast and ERK1/2 phosphorylation in HepG2 cells. In addition to its clinical application to eliminate bacteria, ampicillin could also be used to activate Mpk1 or ERK1/2 in the laboratory.