Objective To investigate the effects and modes of action of combined exposure to black carbon and palmitic acid on mouse spermatogonia GC-1, as well as the role of NRF2 pathway in the damage caused by this combined exposure. Methods A control group, palmitic acid groups(PA), black carbon groups(BC) and combined action groups were set up. Mouse spermatogonia GC-1 cells were treated with PA and BC for 24 h. The cell viability was assessed using by CCK-8 detection method. Real-time quantitative PCR (RT-qPCR) was used to detect the mRNA expression levels of nuclear factor erythroid 2-related factor (NRF2), Kelch-like ECH-related protein 1 (KEAP1), catalase (CAT), heme oxygenase-1 (Hmox1), quinone oxidoreductase 1 (NQO1), glutamyl cysteine ligase (GCLC), autophagy-related genes (Lc3b, P62), and ferroptosis-related genes glutathione peroxidase 4 (GPX4). The content of lipid oxidation (MDA) and reduced glutathione(GSH)were determined by colorimetric method. Results With the increase in exposure concentration, the inhibitory effect of PA and BC on the proliferation of GC-1 cells was enhanced, the IC50 values of PA and BC on GC-1 cells after 24 h were 320μmol/L and 560μg/mL, respectively. When PA was combined with BC, its IC50 for GC-1 cells was lower than that of the single-agent group, indicating a synergistic effect. The results of RT-qPCR showed that after 24 h of PA and BC exposure, both alone and in combination, the expressions of the antioxidant stress-related genes NRF2, KEAP1, CAT, Hmox1, NQO1, GCLC, ferroptosis-related genes GPX4, autophagy-related genes Lc3b and P62 were changed compared with the control group. The expression of NRF2 and Hmox1 was up-regulated (P<0.05), and KEAP1 was down-regulated(P<0.05). The expression of CAT was increased, except in the 20μmol/L PA group and the 40μmol/LPA group (P<0.05). The expression of NQO1 was increased, except in the 20μmol/L PA, 70μg/mL BC and 280μg/mL BC groups (P<0.05). The expression of GCLC was increased, except in the 20μmol/L PA group and the BC group (P<0.05). The expression of ferroptosis-related gene GPX4 was increased, except in the 20μmol/L PA group and the 40μmol/LPA group (P<0.05). The expression of autophagy-related genes Lc3b and P62 was increased, except in the 20μmol/L PA group and the 40μmol/LPA group (P<0.05). The content of MDA in each group was higher than that in the control group (P<0.01),while the content of GSH in each group was lower than that in the control group (P<0.01). Conclusions Both PA and BC can inhibit the proliferation of spermatogonia, and when the two are present simultaneously, they have a synergistic effect and enhance cytotoxicity. The combination of low-level PA and BC exposure to spermatogonia for 24 h triggered an early adaptive response, the mRNA expression level of antioxidant stress-related gene KEAP1 decreased, while the mRNA expression level of NRF2, Hmox1, NQO1, GCLC and CAT increased, the mRNA expression level of ferroptosis-related gene GPX4 increased, the mRNA expression level of autophagy-related genes P62 and Lc3b increased. The content of MDA in cells increased, and the content of GSH decreased. It causes the cells to react to oxidative stress and suggests that the NRF2 pathway may be involved in regulating the process of damage to spermatogonia caused by the combined exposure to PA and BC.