Carbaryl, one of the most important insecticides, is widely produced and used. To explore carbaryl-induced genotoxic effects of spermatozoa, particularly DNA damage and chromosome aberrations (CA), we first examined conventional semen parameters, the progression and motion parameters of the spermatozoa among 16 carbaryl-exposed workers and 30 internal and external control individuals. Sperm DNA damage represented as positive percentage of DNA fragmentation was detected by a modified terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Then numerical CA of chromosome X, Y, and 18 were investigated by multicolor fluorescence in situ hybridization (FISH). The results showed significant differences in the percentage of sperm abnormality between carbaryl-exposed group and the external control group (p = 0.008). Mean (+/-SD) percentage of spermatozoa with fragmented DNA in carbaryl-exposed group (21.04 +/- 8.88%) was significantly higher than those in the internal (13.36 +/- 12.17%) and external control groups (13.92 +/- 7.15%), respectively (p = 0.035 and p = 0.030). Using FISH, we observed the frequency of sperm sex chromosome disomy was 0.661 +/- 0.238% in the exposed group, which was significantly higher than that in the external control group (0.386 +/- 0.140%) (p = 0.001), and the carbaryl-exposed group (0.276 +/- 0.126%) had an elevated chromosome 18 disomy compared with the internal (0.195 +/- 0.094%) and external control groups (0.124 +/- 0.068%), respectively (p < 0.05 and p < 0.01). In addition, carbaryl-exposed donors had significantly higher sperm nullisomic frequencies of sex chromosomes and chromosome 18 than the external controls (p < 0.01) but not the internal controls. In summary, the frequencies of aneuploidy and numerical CA showed significant differences between exposed group and control groups (p < 0.05 and/or p < 0.01). Moreover, positive correlations were found between sex chromosome disomy, aneuploidy rate, and morphologic abnormalities in spermatozoa of all donors (r = 0.564 and r = 0.555, p < 0.01). Our findings suggested that carbaryl might induce morphologic abnormalities and genotoxic defects of spermatozoa among exposed workers by causing DNA fragmentation and numerical CA in spermatogenesis as a potential genotoxicant. The evidence also indicated that the spermatotoxicity induced by carbaryl exposure might be related to adverse reproductive outcomes.