Artificial cells (ACs) aim to mimic selected structural and functional features of mammalian cells. In this context, energy generation is an important challenge to be addressed when self-sustained systems are desired. Here, mitochondria isolated from HepG2 cells are employed as natural subunits that facilitate chemically driven adenosine triphosphate (ATP) synthesis. The successful mitochondria isolation is confirmed by monitoring the preserved inner membrane potential, the respiration, and the ATP production ability. The encapsulation of the isolated mitochondria in gelatin-based hydrogels results in similar initial ATP production compared to mitochondria in solution with a sustained ATP production over 24 h. Furthermore, luciferase is coencapsulated with the mitochondria in gelatin-based particles to create ACs and employ the in situ produced ATP to drive the catalytic conversion of d-luciferin. The coencapsulation of luciferase-loaded liposomes with mitochondria in gelatin-based hydrogels is additionally explored where the encapsulation of mitochondria and liposomes resulted in clustering effects that are likely contributing to the functional performance of the active entities. Taken together, mitochondria show potential in cell mimicry to facilitate energy-dependent processes.