Currently, lung cancer has remained the most common cause of cancer death while non-small cell lung cancer (NSCLC) accounts for the most of all lung cancer cases. Regardless of multiple existing managements, chemotherapy regimens are still the mainstay of treatment for NSCLC, where pemetrexed has shown cytotoxic activity and has increasingly been used, especially for advanced cases. However, chemo-resistance may inhibit clinical efficacy after long-term use. Mechanisms responsible for chemo-resistance to pemetrexed in NSCLC are plethoric but can be separated into two categories to be discussed: tumor cells and their interactions with drugs. Phenomena relevant to tumor cells such as oncogene or oncoprotein alterations, DNA synthesis, DNA repair, and tumor cell biology behavior are discussed, as well as processes associated with drug dynamics, including drug uptake, drug elimination, and antifolate polyglutamylation. This review will focus on clinical trials and the basic biomedical mechanisms of NSCLC treated with pemetrexed and will describe the underlying mechanisms of resistance to facilitate more efficient clinical therapies to treat patients.