Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4(+) T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4(+) T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21(+), IL-4(+), and IL-22(+) CD4(+) T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFN gamma(+) cells were unaffected. CD4(+) T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.