Nemo‐like kinase (Nlk) is related to the mitogen‐activated protein (MAP) kinases and known to regulate signaling pathways involved in osteoblastogenesis. In vitro Nlk suppresses osteoblastogenesis, but the consequences of the Nlk inactivation in the skeleton in vivo are unknown. To study the function of Nlk, Nlk^loxP/loxP mice, where the Nlk exon2 is flanked by lox^P sequences, were mated with mice expressing the Cre recombinase under the control of the paired‐related homeobox gene 1 (Prx1) enhancer (Prx1‐Cre), the Osterix (Osx‐Cre) or the osteocalcin/bone gamma carboxyglutamate protein (Bglap‐Cre) promoter. Prx1‐Cre;Nlk^Δ/Δ mice did not exhibit a skeletal phenotype except for a modest increase in trabecular number and connectivity observed only in 3‐month‐old male mice. Osx‐Cre;Nlk^Δ/Δ male and female mice exhibited an increase in trabecular bone volume secondary to an increased trabecular number at 3 months of age. Bone histomorphometry revealed a decrease in osteoclast number and eroded surface in male mice, and decreased osteoblast number and function in female mice. Expression of osteoprotegerin mRNA was increased in calvarial extracts, explaining the decreased osteoclast and osteoblast number. The conditional deletion of Nlk in mature osteoblasts (Bglap‐Cre;Nlk^Δ/Δ) resulted in no skeletal phenotype in 1‐ to 6‐month‐old male or female mice. In conclusion, when expressed in undifferentiated osteoblasts, Nlk is a negative regulator of skeletal homeostasis possibly by targeting signals that regulate osteoclastogenesis and bone resorption. J. Cell. Physiol. 229: 1736–1743, 2014. © 2014 Wiley Periodicals, Inc.