Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)‐stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV‐2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS‐induced iNOS protein expression and nitric oxide production. Real‐time PCR experiments showed that LPS‐induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS‐stimulated nuclear translocation and phosphorylation of nuclear factor (NF)‐κB as well as the degradation of inhibitor of κB (IκB)‐α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ–NF‐κB activation was found to be dependent on the Ca2+/calcium–calmodulin‐dependent protein kinase II (CaMKII)/transforming growth factor β‐activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca2+ or inhibition of CaMKII reduced LPS‐induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS‐induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca2+]i, and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na+/H+ exchanger 1 (NHE1), which is an important exchanger responsible for Ca2+ influx in LPS‐stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70‐Ca2+‐CaMKII/TAK1–NF‐κB signals in BV‐2 microglia, providing further insight into the functions of Hsp70 in the CNS. © 2015 Wiley Periodicals, Inc.