Laminin is a major structural element of the basal lamina consisting of an α-chain, a β-chain, and a γ-chain arranged in a cross-like structure, with their C-terminal inter-coiled. Laminin is abundantly expressed in the hippocampus of mature brain and is implicated in several psychiatric disorders, but its possible role involved in learning and memory function is not known. This issue was examined here. Our results revealed that water maze training significantly decreased laminin-β1 (LB1) expression in the rat hippocampal CA1 area. Transfection of LB1 WT plasmid to hippocampal CA1 neurons impaired water maze performance in rats. Meanwhile, it decreased the phosphorylation level of ERK/MAPK and protein kinase serum- and glucocorticoid-inducible kinase-1 (SGK1). By contrast, knockdown of endogenous LB1 expression using RNA interference (LB1 siRNA) enhanced water maze performance. Meanwhile, it increased the phosphorylation level of ERK/MAPK and SGK1. The enhancing effect of LB1 siRNA on spatial learning and on the phosphorylation of ERK/MAPK and SGK1 was blocked by co-treatment with the MEK inhibitor U0126 at a concentration that did not apparently affect spatial learning and ERK/MAPK phosphorylation alone. Further, the enhancing effect of LB1 siRNA on spatial learning and SGK1 phosphorylation was similarly blocked by co-transfection with SGK1 siRNA at a concentration that did not markedly affect spatial learning and SGK1 expression alone. These results together indicate that LB1 negatively regulates spatial learning in rats. In addition, LB1 impairs spatial learning through decreased activation of the ERK/MAPK-SGK1 signaling pathway in the rat hippocampus.