Objective To investigate the effects and modes of action of combined black carbon (BC) and palmitic acid ( PA) exposure on mouse spermatogonia GC-1, as well as the role of the nuclear factor erythroid 2-related factor (NRF2) pathway in this process. Methods Control, PA, BC, and combined treatment groups were established. Mouse spermatogonia GC-1 cells were treated with PA and BC for 24 h. Cell viability was assessed using Cell Counting Kit-8 ( CCK-8) detection. Reverse-transcription quantitative PCR ( RT-qPCR) was used to detect mRNA expression levels of antioxidant stress-related genes Nfe2l2, Kelch-like erythroid cell-derived protein with CNC homology(ECH)-related protein 1 (Keap1), catalase (Cat), heme oxygenase-1 (Hmox1), quinone oxidoreductase1 (Nqo1), glutamyl cysteine ligase ( Gclc), autophagy-related genes ( Lc3b and Sqstm1), and ferroptosis-related genes glutathione peroxidase 4 (Gpx4). Lipid oxidation (MDA) and reduced glutathione (GSH) were determined using colorimetry. Western blot was used to detect NRF2 protein expression levels. Results With the increase in exposure concentration, the inhibitory effect of PA and BC on GC-1 proliferation was enhanced; half maximal inhibitory concentration(IC₅₀) values of PA and BC on GC-1 cells after 24 h were 320 μmol / L and 560 μg / mL,respectively. When PA was combined with BC, its IC₅₀ for GC-1 cells was lower than that of the single-agent group,indicating synergy. RT-qPCR showed that after 24 h of PA and BC exposure, both alone and in combination, the expression of Nfe2l2, Keap1, Cat, Hmox1, Nqo1, Gclc, Gpx4, Lc3b, and Sqstm1 differed compared with the control group (P<0. 05). MDA content in each experimental group was higher than that in the control group (P<0. 05),while GSH content was lower (P<0. 05). Western blot showed that NRF2 protein expression increased with increasing PA and BC concentrations compared with the control ( P<0. 05). Conclusions PA and BC both inhibited spermatogonia proliferation, and can synergize when present together to enhance cytotoxicity. When low-level PA and BC are combined and applied to spermatogonia, NRF2 signaling is activated, changing expression levels of oxidative stress, ferroptosis, and autophagy-related genes; this causes cells to react to oxidative stress and suggests that the NRF2 pathway may be involved in regulating spermatogonia damage caused by combined exposure to PA and BC.