Lilies are well-known flowers with large anthers and a high quantity of pollen that easily contaminates clothing and tepals. The anthers need to be artificially removed, leading to production problems. Cultivating male-sterile or pollen-free lilies could solve these problems. The key period of male sterility in a specific male-sterile hybrid lily population was determined through cytological observation. The contents of hormones, soluble sugar, soluble protein, and proline were determined by high-performance liquid chromatography, tandem mass spectrometry and colorimetry. Transcriptome sequencing was used to identify the genes with altered expression. The key period of male sterility was determined to be the microspore mother and tetrad stages. The hormone contents were abnormal in the sterile line compared with the fertile line. The indole-3-acetic acid (IAA) content was higher in the sterile line than in the fertile line at all stages, while the gibberellic acid 4 (GA4) content showed the opposite result. Abscisic acid (ABA) accumulated in the sterile line in both the microspore mother and tetrad stages, and the zeatin riboside (ZR) content in the sterile line increased at the microspore mother stage but decreased at the tetrad stage. The contents of soluble sugar, soluble protein and proline were higher in the fertile line than in the sterile line. Genes involved in auxin and ABA synthesis and signalling pathways were highly expressed in the male-sterile line. Our data suggested that abnormal contents of hormones in the microspore mother and tetrad stages resulted in pollen abortion in a male-sterile hybrid lily population, which indicated that the hormone balance in specific stages plays critical functions in pollen development in lilies.