Single-walled carbon nanotubes (SWCNTs) are widely utilized for industrial, biomedical, and environmental purposes. The toxicity of Carboxylated SWCNTs (SWCNTs−COOH) in in vivo models, particularly Caenorhabditis elegans (C. elegans), and in vitro human cells is still unclear. In this study, C. elegans was used to study the effects of SWCNTs−COOH on lethality, lifespan, growth, reproduction, locomotion, reactive oxygen species (ROS) generation, and the antioxidant system. Our data show that exposure to ≥1 μg·L−1 SWCNTs−COOH could induce toxicity in nematodes that affects lifespan, growth, reproduction, and locomotion behavior. Moreover, the exposure of nematodes to SWCNTs−COOH induced ROS generation and the alteration of antioxidant gene expression. SWCNTs−COOH induced nanotoxic effects at low dose of 0.100 or 1.00 μg·L−1, particularly for the expression of antioxidants (SOD-3, CTL-2 and CYP-35A2). Similar nanotoxic effects were found in human cells. A low dose of SWCNTs−COOH induced ROS generation and increased the expression of catalase, MnSOD, CuZnSOD, and SOD-2 mRNA but decreased the expression of GPX-2 and GPX-3 mRNA in human monocytes. These findings reveal that background-level SWCNTs−COOH exerts obvious adverse effects, and C. elegans is a sensitive in vivo model that can be used for the biological evaluation of the toxicity of nanomaterials.