Purpose Methotrexate is one of the widely used agents to treat various solid tumors, although its usage brings some challenges because of its poor bioavailability and/or dose-dependent side effects. Increasing the solubility, permeability, and consequently bioavailability in company with minimizing side effects are always desired to formulate such these drugs. In this paper, we concentrated on formulation parameters of methotrexate-loaded niosomes, in an attempt to facilitate its solubility. Methods Various formulation parameters were utilized, including surfactant types, the amount of membrane additives, and types of hydrating agent. Methotrexate-loaded niosomes were fully characterized. The cell viability and hemolysis assay were evaluated. The effects of developed formulation on cellular interaction, growing, and micronuclei assay were also investigated. Results Methotrexate (MTX)-loaded niosomes were produced in small particle size (241.3 +/- 65.7 nm) with high uniformity using sodium hydroxide as hydrating solution. Their entrapment efficiency was 59.5 +/- 3.0% and the release completed in 6 h. The incorporation of methotrexate into niosomes was confirmed, and their higher efficiency on cell viability was presented. MTX-loaded niosomes were found safe and acceptable for parenteral administration. The number of apoptotic and dead cells were increased significantly in all types of cells treated with niosomes. The apoptosis-mediated cell death was triggered by produced niosomes. Conclusion This study suggests that MTX-loaded niosomes formulated with sodium hydroxide provided a significant improvement in niosome characteristics with the help of solubility enhancement strategies. The cellular assessments proved its potential efficacy. It is concluded that these promising results should be promoted in further studies.