Summary Recently, the question of whether vitamin D exerts an effect on the pathogenic process of infertility has become the centre of attention. There are some controversial conclusions on this issue. Based on previous studies, we sought to explore the difference of serum 25‐hydroxyvitamin D3, 1,25‐dihydroxyvitamin D3 levels between infertile patients and fertile men, and to find the influence on semen quality. The analysis of serum 25‐hydroxyvitamin D3 level showed no significant difference between infertile patients and fertile men. However, the levels of serum 1,25‐dihydroxyvitamin D3 in oligospermia (P < 0.05), asthenospermia (P < 0.01), oligoasthenospermia (P < 0.05) and azoospermia (P < 0.01) patients were significantly lower than those in fertile men. Moreover, serum 1,25‐dihydroxyvitamin D3 level was positively correlated with progressive motility and total sperm number in infertile patients. In addition, a positive correlation between serum prolactin and 1,25‐dihydroxyvitamin D3 was observed in fertile men. Our results indicated that lower vitamin D could be a risk factor for poor semen quality in infertile men. The 1,25‐dihydroxyvitamin D3, as the biologically active form of vitamin D, may be more significant.