Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet. Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h). Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.