Research background: The use of artificial intelligence represents a modern approach to improving the strategic management of enterprises. Several studies have confirmed that gender plays a key role in the context of sustainable growth within the business environment. Purpose of the article: The aim of this article was to evaluate the extent to which artificial intelligence is utilized in medium and large companies operating in the business environment of the Czech Republic. Methods: A questionnaire was used to survey HR managers regarding the use of artificial intelligence in medium and large enterprises. A total of 451 HR managers from the business sector participated in the study. The research was conducted in 2024 in the Czech Republic. The assumption of normal distribution was tested using the Kolmogorov–Smirnov test, and homoscedasticity was evaluated using Levene’s test. Statistical hypotheses were verified using auxiliary statistics and the non-parametric Mann–Whitney U test. Findings & Value added: The empirical results revealed several interesting findings. Statistically significant gender-based differences were observed in HR managers’ evaluations of artificial intelligence use. Male HR managers assessed AI applications more positively than their female counterparts, particularly in areas such as automation of administrative tasks, recruitment and selection, process efficiency, support for CSR report preparation, and management. However, no significant gender differences were found in the evaluation of AI's impact on improving the employee experience.