Soil nitrogen activation (N_act) is pivotal for the global N cycle, influencing crop N availability and environmental N losses. However, it remains unclear how climate and fertilization individually, or jointly, affect soil N_act over multiple decades. Here, we examined the dynamic shifts in N_act between the first decade and later period in soils treated with non-fertilizer, mineral fertilizer with/without manure, mineral fertilizer with stover return, in 1982 and 1990, in six typical agricultural zones. Results revealed that soil total N (TN) and available N (AN) increased at rates of 10.1–58.2 and 1.41–4.13 mg kg^–1 yr^–1, respectively, by manure application at five sites, suggesting that manure enhanced both soil N storage and availability. The GZL site exhibited the highest annual change rate (ACR) in TN (48.3–58.2 mg kg^–1 yr^–1), while the ZY site had the lowest (10.1 mg kg^–1 yr^–1). Conversely, the YL site showed the highest ACR in AN (3.65–4.13 mg kg^–1 yr^–1), whereas the ZZ site exhibited the lowest (1.41–2.23 mg kg^–1 yr^–1). Notably, the N_act rates with manure application were higher in the first decade (42–181 mg g^–1) than in the later period (33–92 mg g^–1) at all sites. Overall, the average N_act of all treatments in the first decade (79–105 mg g^–1) across six study sites was higher than in the later period (30–78 mg g^–1). Variance portioning analysis indicated that soil properties’ contribution to N_act increased from 35% to 45% over time, while climatic conditions’ effect decreased from 19% to 8%. Structural equation modeling confirmed a direct impact of annual temperature on N_act, with path coefficients of 0.86 in the first decade and 0.45 in the later period. These results show that the impacts of climate on soil N_act were attenuated under long-term fertilizer application, while the interactions between climate and soil had an enhanced impact on N_act in the later period of fertilization. This context-specific insight can guide soil management strategies to enhance N availability, modulate the effects of climate change on agricultural production, and minimize environmental N losses.