This study aims to explore the formulation and preparation process of aloperine-based thermosensitive gels and investigates their preliminary pharmacodynamics and mechanisms of spermicidal action through antifertility, vaginal irritation, and anti-inflammatory experiments. The minimum spermicidal concentration of aloperine was determined via in vitro spermicide assays. The calcium ion probe Fluo-4AM, combined with flow cytometry, was employed to measure intracellular calcium ion concentrations in mouse sperm, while q-PCR technology was utilized to analyze the expression levels of the Catsper gene, a key regulator of calcium channels in mouse sperm. Poloxamer 407 (P407) and poloxamer 188 (P188) were selected as thermosensitive matrix materials, with gelling temperature (Tgel) serving as the primary evaluation criterion. A single-factor screening was performed to identify suitable gel matrices, and an orthogonal design methodology was employed to optimize the formulation and preparation process. Subsequently, Tgel, gelling time, pH, and viscosity of the optimized aloperine thermosensitive gel were thoroughly characterized. The anti-fertility effects and vaginal irritation of the gel through intravaginal administration were systematically investigated, along with its anti-inflammatory efficacy against xylene-induced ear edema in mice. The results demonstrated that aloperine exhibits a minimum spermicidal concentration of 25 mg/mL under acidic conditions. Compared with the normal saline control group, the aloperine-treated group displayed a significant reduction in intracellular calcium ion concentrations in sperm. It is hypothesized that low concentrations of aloperine suppress intracellular calcium levels by modulating the expression of the Catsper gene. The optimized gel formulation comprises 160 mg/g P407, 40 mg/g P188, 25 mg/g propylene glycol, 25 mg/g aloperine, 78.59 mg/g lactic acid, and 100.80 mg/g sodium lactate. The prepared gel exhibits an average Tgel of 33 ℃, a gelling time of 97.67 s, a pH value of 4.19, and a viscosity of 199.50 cP at 25 ℃. At a concentration of 25 mg/g, the aloperine gel achieved a 100% contraceptive efficacy rate. Vaginal irritation testing revealed no significant adverse effects, and the gel markedly attenuated xylene-induced auricular swelling in mice. In conclusion, this study suggests that aloperine may inhibit mouse sperm motility by attenuating intracellular calcium ion influx, potentially through the modulation of Catsper gene expression. The aloperine thermosensitive gel, with its simple preparation process, strong temperature sensitivity, ease of administration, and high efficacy, demonstrates substantial potential for further research and practical application.