Effects of ultraviolet A (UVA) and ultraviolet B (UVB) on mitochondrial function and membrane integrity of sea urchin sperm were investigated using flow cytometry and fluorescent probes. Both UVA and UVB impaired sperm mitochondrial function in a dose-dependent manner. Covariance analysis further showed that the slopes of change in mitochondrial function in relation to UVA and UVB were significantly different, suggesting that the modes of action were different. UVA did not affect membrane integrity, while membrane integrity showed a linear reduction with increasing UVB doses. Sperm mitochondria function showed significant positive correlations with sperm motility and subsequent fertilization success. Overall, our results showed that both UVA and UVB could decrease sperm motility and fertilization success through impairment of mitochondrial function, whereas UVB alone could cause additional damage through impairing the functional integrity of sperm membrane. Mitochondrial function of sperm may also offer a reliable ecotoxicological biomarker for predicting fertilization success in urchins.