This study investigated the effects of different temperatures, photoperiods and spermidine concentrations on the flowering time regulation of Anoectochilus roxburghii by measuring changes in the soluble sugar, soluble protein, malondialdehyde and proline contents, and the peroxidase, superoxide dismutase and catalase activities in A. roxburghii flower buds. The flowering time could be advanced under 25/20 °C (day/night), 16/8-h (day/night) long day conditions or low spermidine concentrations. The plants grew more rapidly and flowering rates were greater. The flowering time could be delayed under a low temperature of 20/15 °C or 8/16-h short day conditions, resulting in a low flowering rate. Under a high temperature of 30/25 °C or high spermidine concentrations, the plants could not flower normally and even died. There were significant differences in the seven measured indices among the various treatments. Thus, different treatments had significant effects on the flowering time regulation and flowering quality of A. roxburghii, providing a reliable theoretical basis for further studies on the flowering-related regulatory mechanisms of A. roxburghii.