The Negev Highlands arid region (southern Levant) shows evidence of sharp settlement fluctuations, with several periods of strong human activity separated by centuries with no evidence of sedentary life. In this study, we used the palynological method in order to shed light on the region’s demographic history in the Bronze and Iron Ages. Fifty-four samples of pollen were collected and analyzed from secure archaeological contexts in four Negev Highlands sites: Nahal Boqer 66, dated to the Early Bronze Age and Early Intermediate Bronze Age (ca. 3200–2200 BCE); Ein Ziq, dated to the Early Intermediate Bronze Age (ca. 2500–2200 BCE); Mashabe Sade, dated to the Intermediate Bronze Age (ca. 2500–2000 BCE); and Haroa, dated to the Iron Age IIA (ca. late 10th through 9th centuries BCE). Our study revealed no evidence of cereal cultivation, with some hints that the inhabitants’ diets may have included plants gathered from the wild. Only one of the sites, Nahal Boqer 66, showed micro-indicators of animal dung remains, suggesting that the inhabitants herded animals. The palynological evidence did, however, emphasize that the livestock there were not fed or supplemented with agricultural by-products but rather grazed freely on wild vegetation. The pollen data also suggest that all four sites were occupied only during late winter and spring. The activity in the Negev Highlands during the third millennium BCE was probably related to the copper industry in the Arabah and to copper transportation to settled neighboring lands, especially Egypt. A relatively humid climate supported the trade through the Negev Highlands. Deterioration in both climate conditions and settlement activity was documented in the second half of the Intermediate Bronze Age.