Autophagy is a catabolic lysosomal-dependent pathway involved in the degradation of cellular materials, supplying precursor compounds and energy for macromolecule synthesis and metabolic needs [1,2]. There are three distinct forms of autophagy, including macroautophagy (simply referred to here as autophagy), microautophagy and chaperonemediated autophagy (CMA) [1]. All three are essential in regulating several key biological processes including cell death, metabolism, stress responses, as well as immune surveillance and disease development [2,3]. At early stages of tumorigenesis, autophagy is reduced, favoring cancer formation by increasing DNA damage. However, at later stages and in established tumor cells, autophagy is usually reactivated as an adaptive response to stressful conditions, helping to sustain cancer stem cells (CSCs) and their tumorigenic potential and contributing overall to resistance to cancer treatments [4]. As autophagy has both positive and negative roles in cancer, both its inhibition and activation may have therapeutic benefits for cancer patients. Despite making much progress in understanding the molecular mechanisms of autophagy in cancer, many questions remain concerning its role in cancer development and treatment. This Special Issue of Cells, entitled “molecular connections between autophagy, programmed cell death pathways and differentiation in cancer cells”, presents some of the latest findings on the role of autophagy in regulated cell death pathways, metabolism, and immune responses, in addition to showcasing its involvement in cancer development, the maintenance of CSCs and and resistance to cancer therapy. This issue features six review articles and eight original studies.